Illumination device for spatial and temporal control of morphogen signaling in cell cultures

ABSTRACT

Provided are systems and methods for spatially and temporally controlling light with an illumination device comprising a light source operably connected to a circuit board, one or more light guide plates, one or more optical masks, a controller, and a computer readable medium, comprising instructions that, when executed by the controller, cause the controller to: illuminate a cell or a substrate with light from the light source, and spatially and temporally control illumination of light to the cell or the substrate with one or more illumination parameters, wherein the one or more light guide plates provides uniform illumination of the light. Also provided herein are methods of screening using the system and/or device of the present disclosure.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/847,684, filed May 14, 2019, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NS087253 awardedby the National Institutes of Health. The government has certain rightsin the invention.

INTRODUCTION

Morphogen gradients are present throughout development and orchestratethe dynamic, coordinated movement and differentiation of cellpopulations. Spatially and temporally varying patterns of morphogenslocalize signaling to specific subpopulations of cells. Geneticperturbation and biomolecular treatment with pathway agonists orinhibitors have given immense insight into the key regulators ofdevelopmental progression, yet spatially-varying interactions betweencell subpopulations and time-varying signal dynamics and thresholdsremain largely unstudied, since such patterns of signaling are difficultto perturb and control in model developmental systems.

There is a need for optogenetic tools that control spatial and temporalpatterns of light for high-throughput optogenetic screening.

SUMMARY

Provided are systems and methods for spatially and temporallycontrolling light with an illumination device comprising a light sourceoperably connected to a circuit board, one or more light guide plates,one or more optical masks, a controller, and a computer readable medium,comprising instructions that, when executed by the controller, cause thecontroller to: illuminate a cell or a substrate with light from thelight source, and spatially and temporally control illumination of lightto the cell or the substrate with one or more illumination parameters,wherein the one or more light guide plates provides uniform illuminationof the light. Also provided herein are methods of screening using thesystem and/or device of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Overview of illumination device, LAVA, for optogeneticstimulation of hESC cultures.

FIGS. 2A-2C. Optical design for illumination uniformity of tissueculture wells.

FIGS. 3A-3D. Optogenetic induction of Bra expression is light-doseresponsive.

FIGS. 4A-4D. Characterization of temporal control using LAVA devices.

FIGS. 5A-5F. OptoWnt induces epithelial to mesenchymal transition andprimitive streak-like behavior.

FIG. 6. System block diagram of LAVA device.

FIG. 7. Emission spectrum of 470 nm blue LEDs matches absorptionspectrum of Cry2. Cry2 spectrum adapted from reference.

FIGS. 8A-8B. Screenshot of GUI for illumination device control. User caninput parameters for desired intensities, blinking sequences, ortemporal functions for each individual well and upload settingswirelessly to the device.

FIGS. 9A-9H. Validation of Zemax ray tracing model.

FIGS. 10A-10E. Results of Zemax modeling at variable light guidethicknesses, d₁ and d₂.

FIG. 11. Coefficient of variation of light intensity between the 24independent light channels measured at different programmed intensities.Green points correspond to optical configuration with d=1 cm, violetpoints show optical configuration with d=1.5 cm.

FIGS. 12A-12B. FIG. 12A) Immunostaining quantification for average Braintensity per hESC in response to increasing light intensity after 24 hrillumination or 3 μM CHIR treatment. Graph shows mean±1 s.d., n=3replicates. FIG. 12B) How cytometry histograms of optoWnt hESCsexpressing eGFP reporter for Bra after 24 hr illumination at varyinglight intensities. Graph shows sum of n=3 replicates.

FIGS. 13A-13D. Phototoxicity during continuous optogenetic stimulationof hESC cultures.

FIG. 14. Illumination power meter measurements of programmed blinkingsequences show signal inaccuracy at 1 ms pulses. Voltage signal frompower meter measured with oscilloscope and is proportional toirradiance.

FIGS. 15A-15C. FIG. 15A) Images of adhesive die-cut masks applied usingtransfer tape (top) onto 24-well cell culture plate (bottom). FIG. 15B)Brightfield images of die-cut mask illustrate resolution limit ofcutter. Scale bar 3 mm FIG. 15C) Schematic of light scattering fromphotomask.

FIG. 16. Screenshot of Zemax model parameters of LAVA well, optimizedfor uniform 24-well illumination.

FIGS. 17A-17B. Circuit board layout (top) and schematic (bottom) for24-well LAVA device, PCB1.

FIGS. 18A-18B. Circuit board layout (top) and schematic (bottom) forLAVA device power distribution, PCB2.

FIGS. 19A-19B. Circuit board layout (top) and schematic (bottom) for96-well LAVA device, PCB1.

DEFINITIONS

A “light guide plate” as used herein in its conventional sense, refersto a structure or material in which one or more light guides arepositioned or formed therein.

A “light guide” as used herein in its conventional sense, refers to astructure or material that transmits illumination from a light source.

An “optical mask” as used herein in its conventional sense, refers to asubstrate or material that selectively blocks a wavelength of light.However, an optical mask may have a region on the material in whichlight can pass through (e.g. aperture, core region, cut-out feature,etched feature).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the opticalmask” includes reference to one or more optical masks and equivalentsthereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

Provided are systems, devices, and methods for spatially and temporallycontrolling light with an illumination device comprising a light sourceoperably connected to a circuit board, one or more light guide plates,one or more optical masks, a controller, and a computer readable medium,comprising instructions that, when executed by the controller, cause thecontroller to: illuminate a cell or a substrate with a light-basedactivation signal (e.g. light) from the light source, and spatially andtemporally control illumination of light to the cell or the substratewith one or more illumination parameters, wherein the one or more lightguide plates provides uniform illumination of the light. Also providedherein are methods of screening using the system and/or device of thepresent disclosure.

Systems and Devices

The present disclosure provides illumination devices and systems forspatially and temporally controlling light. According to aspects of thepresent disclosure, the illumination device includes a light sourceoperably connected to a circuit board and configured to produce light;one or more light guide plates comprising one or more light guides; oneor more optical masks positioned on a surface of one or more wells of atissue culture plate; a controller; and a computer readable medium, thatincludes instructions that, when executed by the controller, cause thecontroller to illuminate the cell or the substrate with light from thelight source; and spatially and temporally control illumination of lightto the cell or the substrate in the one or more wells with one or moreillumination parameters. In some aspects, the one or more light guidesis configured to provide uniform illumination of the light in the one ormore wells of the tissue culture plate. In some aspects, theillumination device is connected to a tissue culture plate comprising acell or a substrate in one or more wells of the tissue culture plate.

Tissue Culture Plate

In some aspects, the illumination device is positioned adjacent to aculture plate. In some cases, the culture plate is a tissue cultureplate (e.g. a cell culture plate or a multi-well plate). In some cases,the illumination device is reversibly connected to a tissue cultureplate. By “adjacent”, as used herein in its conventional sense to referto be connected, linked, fastened, or positioned on a surface of theillumination device. In some cases, an illumination device positionedadjacent to a culture plate includes a gap or space between the tissueculture plate and the illumination device.

In some cases, the tissue culture plate includes one or more wells. Insome cases, the tissue culture plate includes 24 wells. In some cases,the tissue culture plate includes 96 wells. In some cases, the tissueculture plate includes 384 wells.

In some cases, the tissue culture plate is made from an opaque polymer.In some cases, the tissue culture plate is made from a black polymer. Insome cases, the tissue culture plate is made from a material thatprevents light from bleeding through between the one or more wells.

In some cases, the tissue culture plate includes a coverglass bottom. Insome cases, the one or more wells include a coverglass bottom. In somecases, the coverglass bottom has a thickness ranging from 150-200 μm. Insome cases, the coverglass bottom has a thickness of about 150 μm, 160μm, 170 μm, 180 μm, 190 μm, or 200 μm.

In some cases, the illumination device positioned adjacent to the tissueculture plate is configured to be placed in an incubator. In some cases,the illumination device positioned adjacent to the tissue culture plateplaced in an incubator can be controlled wirelessly (e.g. controlledwirelessly without removing the illumination device positioned adjacentto the tissue culture plate from the incubator.

Light Source

In some cases, the illumination device includes a light source. In somecases, the light source is configured to product a light-basedactivation signal (e.g. light). In some cases, the light source isconfigured to be positioned adjacent to a circuit board. In some cases,the light source is operably adjacent to a circuit board. In some cases,the light source is configured to be connected to the circuit board.

In some cases, the light source can be a light emitting diode (LED). Insome cases, the light source includes one or more LEDs. In some cases,the LED can generate white, blue, red, and/or green light. In somecases, the LED can generate amber and/or yellow light. In some cases,the LEDs are micro LEDs. In some cases, the LEDs are embedded into acircular array of the circuit board. In some embodiments, the lightsource is a solid state laser diode or any other means capable ofgenerating light. The light generating means can generate light havingan intensity sufficient to activate a cell, protein, and/or a substrate.In some cases, the light includes an irradiance (e.g. light afterpassing through the optics) having an intensity of any of about 0.005μW/mm², 0.006 μW/mm², 0.0007 μW/mm², 0.008 μW/mm², 0.009 μW/mm², 0.01μW/mm², 0.02 μW/mm², 0.03 μW/mm², 0.04 μW/mm², 0.05 μW/mm², 0.1 μW/mm²,0.2 μW/mm², 0.3 μW/mm², 0.4 μW/mm², 0.5 μW/mm², about 0.6 μW/mm², about0.7 μW/mm², about 0.8 μW/mm², about 0.9 μW/mm², about 1.0 μW/mm², about1.1 μW/mm², about 1.2 μW/mm², about 1.3 μW/mm², about 1.4 μW/mm², about1.5 μW/mm², about 1.6 μW/mm², about 1.7 μW/mm², about 1.8 μW/mm², about1.9 μW/mm², about 2.0 μW/mm², about 2.1 μW/mm², about 2.2 μW/mm², about2.3 μW/mm², about 2.4 μW/mm², about 2.5 μW/mm², about 3 μW/mm², about3.5 μW/mm², about 4 μW/mm², about 4.5 μW/mm², about 5 μW/mm², about 5.5μW/mm², about 6 μW/mm², about 7 μW/mm², about 8 μW/mm², about 9 μW/mm²,about 10 μW/mm², about 11 μW/mm², about 12 μW/mm², about 13 μW/mm²,about 14 μW/mm², about 15 μW/mm², about 16 μW/mm², about 17 μW/mm²,about 18 μW/mm², about 19 μW/mm², about 20 μW/mm², about 21 μW/mm²,about 22 μW/mm², about 23 μW/mm², about 24 μW/mm², or about 25 μW/mm²,inclusive, including values in between these numbers. In some cases, thelight includes an irradiance having an intensity ranging from about0.0001 to about 25 μW/mm², about 25 to 50 μW/mm², about 50-100 μW/mm²,about 100-150 μW/mm², or 150-200 μW/mm². In other embodiments, thelight-generating means produces light having a frequency of at leastabout 100 Hz. In some cases, the light source produces light having anintensity of any of about 0.05 mW/mm², 0.1 mW/mm², 0.2 mW/mm², 0.3mW/mm², 0.4 mW/mm², 0.5 mW/mm², about 0.6 mW/mm², about 0.7 mW/mm²,about 0.8 mW/mm², about 0.9 mW/mm², about 1.0 mW/mm², about 1.1 mW/mm²,about 1.2 mW/mm², about 1.3 mW/mm², about 1.4 mW/mm², about 1.5 mW/mm²,about 1.6 mW/mm², about 1.7 mW/mm², about 1.8 mW/mm², about 1.9 mW/mm²,about 2.0 mW/mm², about 2.1 mW/mm², about 2.2 mW/mm², about 2.3 mW/mm²,about 2.4 mW/mm², about 2.5 mW/mm², about 3 mW/mm², about 3.5 mW/mm²,about 4 mW/mm², about 4.5 mW/mm², about 5 mW/mm², about 5.5 mW/mm²,about 6 mW/mm², about 7 mW/mm², about 8 mW/mm², about 9 mW/mm², about 10mW/mm², about 11 mW/mm², about 12 mW/mm², about 13 mW/mm², about 14mW/mm², about 15 mW/mm², about 16 mW/mm², about 17 mW/mm², about 18mW/mm², about 19 mW/mm², about 20 mW/mm², about 21 mW/mm², about 22mW/mm², about 23 mW/mm², about 24 mW/mm², or about 25 mW/mm², inclusive,including values in between these numbers.

In some aspects, the light source can be externally activated by acontroller. In some cases, the controller includes a processor. In somecases, the controller can include a power source which can be mounted toa transmitting coil. In some embodiments of the controller, a batterycan be connected to the power source, for providing power thereto. Aswitch can be connected to the power generator, allowing an individualto manually activate or deactivate the power source.

In some cases, the controller is configured to independently illuminateeach of the one or more wells of the tissue culture plate. In somecases, the one or more wells is independently illuminated by the one ormore LEDs.

Circuit Board

In some aspects, the illumination device includes one or more circuitboards. In some cases, the light source is connected to the circuitboard. In some cases, the light source includes one or more LEDs. Insome cases, the circuit board is a printed circuit board (PCB). In somecases, the circuit board includes one or more circular arrays. In somecases, the illumination device includes a first circuit board (PCB1). Insome cases, the PCB1 includes electronics for LED control. In somecases, the illumination device further comprises a power distributionboard. In some cases, the illumination device includes a second circuitboard (PCB2). In some cases, the PCB2 is a power distribution board. Insome cases, the PCB1 contains solder pads for a circular array of 5 LEDsin order to emit light from the 5 LEDs to one well of the tissue cultureplate (e.g. a 24 well tissue culture plate). In some cases, the 5 LEDsare positioned adjacent (e.g. connected to) to the circular array of thecircuit board in series.

In some cases, the one or more LEDs (e.g. two or more, three or more,four or more, five or more, six or more, seven of more, eight or more,nine or more, or ten or more) are symmetrically and radially distributedon one or more circular arrays on the circuit board. In some cases, eachof the one or more circular arrays has a radius ranging from about 2-10mm (e g 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm). Insome cases, each of the one or more circular arrays has a radius ofabout 5 mm.

In some cases, the circuit board includes 5 LEDs symmetrically andradially distributed on each of the one or more circular arrays of thecircuit board. In some cases, the circuit board includes 24 circulararrays. In some cases, the one or more circular arrays is positionedbelow the one or more wells of the tissue culture plate. In some cases,one or more LEDs (e.g. two or more, three or more, four or more, five ormore, seven or more, eight or more, nine or more, or ten or more) oneach of the one or more circular arrays are configured to illuminate onewell of the one or more wells in the tissue culture plate. In somecases, the 5 LEDs on each of the one or more circular arrays areconfigured to illuminate one well of the one or more wells in the tissueculture plate. In some cases, five or more LEDs on each of the one ormore circular arrays are configured to illuminate one well of the one ormore wells in the tissue culture plate.

In some cases, the circuit board includes 1 LED symmetrically andradially distributed on each of the one or more circular arrays of thecircuit board. In some cases, the circuit board includes 96 circulararrays. In some cases, the 1 LED is positioned at approximately thecenter of each circular array.

In some cases, the one or more LEDs on the circuit board are positionedbelow the one or more wells of the tissue culture plate.

In some aspects, the illumination device includes a heat sink mounted onthe circuit board. In some cases, the heat sink is mounted on thecircuit board with a thermally conductive adhesive. In some cases, theheat sink is mounted onto the bottom surface of the first circuit board(e.g. PCB1). In some cases, the heat sink is mounted using a thermallyconductive adhesive. In some cases, the thermally conductive adhesive isArtic Silver, ASTA-7G. In some cases, the heat sink is mounted onto thecircuit board (e.g. first circuit board) in a region without silk screenand thermally conductive electrical vias that draw heat away from theone or more LEDs.

In some cases, the illumination device includes a cooling fan. In somecases, the illumination device includes one or more cooling fans. Insome cases, the illumination device includes two cooling fans. In somecases, the illumination device includes three cooling fans. In somecases, the one or more cooling fans is positioned on the outer edges ofthe circuit board. In some cases, the first circuit board includesheaders for electrical connection to the one or more cooling fans.

In some aspects, the illumination device is connected to a power supply.In some cases, the illumination device is operably connected to thepower supply. In some cases, the illumination device is electricallyconnected to the power supply. In some cases, the power supply connectsto the second circuit board (e.g. PCB2) of the illumination device. Insome cases, power is supplied to the one or more cooling fans and thecontroller through switching voltage regulators.

Controller

In some aspects, the illumination device includes a controller. In somecases, the controller is configured to independently illuminate each ofthe one or more wells.

In some cases, the controller is a microcontroller. In some cases, thecontroller is a Raspberry Pi microcontroller. In some cases, the tissueculture plate is mounted in a position on the illumination device suchthat the tissue culture plate is illuminated from the bottom.

In some cases, the controller further includes one or more LED drivers.In some cases, the LED driver includes one or more channels. In somecases, the LED driver is a 24-channel LED driver. In some cases, the LEDdriver is a 96-channel LED driver. In some cases, the LED driver is a384-channel LED driver. In some cases, the illumination device includestwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, or ten or more LED drivers.In some cases, the illumination device includes four LED drivers. Insome cases, the controller provides for independent control of each ofthe channels of the LED driver (e.g. a 24-channel LED driver, a 96channel LED driver, or a 384-channel LED driver). In some cases, thecontroller is configured to independently illuminate each of the one ormore wells of the tissue culture plate.

In some cases, the device is connected to a power supply. In some cases,the power supply is connected to the LED driver of the illuminationdevice. In some cases, the power supply connects to the second circuitboard through a barrel power jack to power the one or more LEDs throughthe LED driver.

In some cases, the LED driver is electrically connected to the circuitboard. In some cases, the LED driver is mounted and electricallyconnected to the first circuit board of the illumination device. In somecases, the microcontroller is mounted and electrically connected to thefirst circuit board. In some cases, the second circuit board (e.g. PCB2)is mounted and electrically connected to the first circuit board (PCB1).

In some cases, the LED driver is a 24-channel LED driver. The LED driveris configured to control the one or more LEDs. In a non-limitingexample, for 24-well illumination (e.g. illumination of 24 wells in a 24tissue culture plate), a first circuit board includes 24 circular arrayscontaining 5 LEDs radially and symmetrically distributed on each of the24 circular arrays. In some cases, the 24-channel LED driver canindependently control illumination of each well in the 24 tissue cultureplate, wherein each channel of the LED driver controls one well in the24 well plate. Another non-limiting example includes 96-wellillumination (e.g. illumination of 96 wells in a 96 tissue cultureplate), a first circuit board includes 96 circular arrays containing 1LED radially and symmetrically distributed on each of the 96 circulararrays. In some cases, the illumination device can illuminate 24independent channels, wherein each channel controls 4 wells of the 96well plate. In some cases, the illumination device includes four LEDdrivers, each containing 24 channels. In such cases, the four LEDdrivers, combined, include 96 independent channels, wherein each channelcontrols one well of the 96 wells in the tissue culture plate. In somecases, the four LED drivers are operatively connected together toprovide independent control of each well in the 96 wells of the tissueculture plate.

In some cases, the illumination device positioned adjacent to the tissueculture plate is mounted onto a material through vibration-dampeningmounts. A non-limiting example of the material may include an acrylic,plastic, metal, or composite material or any material that is secureenough to constitute a base. In some cases, the acrylic material is anacrylic laser-cut base. In some cases, the vibration-dampening mountsinclude rubber footpegs. In some cases, the vibration-dampening mountsare configured to reduce static or electrical shorting with the tissueculture incubation racks.

Light Guide Plates

Aspects of the present disclosure include an illumination deviceincluding one or more light guide plates. In some cases, the light guideplates include one or more light guides. In some cases, the one or morelight guides are configured to produce a spatial pattern. By “arc oflight”, as used herein, refers to the shape of the spatial pattern thatis cut in the optical mask, used to illuminate the sample (e.g. cell orsubstrate). In some cases, the shape of the spatial pattern is a curvedline (e.g., an arc) cut in the optical mask. In some cases, the one ormore light guide plates comprises a first light guide plate and a secondlight guide plate. In some cases, the first light guide and the secondlight guide can include light guides that are the same form of eachother (e.g. made from the same material, and/or have the samedimensions, etc.). In some cases, the first light guide and the secondlight guide can include light guides that are different forms of eachother (e.g. made from different materials, and/or have differentdimensions, etc.)

In some cases, the one or more light guide plates include one or morelight guides. In some cases, the one or more light guides comprises 24light guides, 96 light guides, or 384 light guides. In some cases, theillumination device has one or more light guides within each of the oneor more light guide plates. For example, in some cases, the light guideswithin each of the one or more light guide plates are held in an arrayby a surrounding frame of the light guide plate. In some cases, theillumination device has 24 light guides held in an array by asurrounding frame of the light guide plate. In some cases, theillumination device has 96 light guides held in an array by asurrounding frame of the light guide plate. In some cases, theillumination device has 384 light guides held in an array by asurrounding frame of the light guide plate.

In some cases, the one or more light guides has a diameter of about 5 ormore mm, about 6 or more mm, about 7 or more mm, about 8 or more mm,about 9 or more mm, about 10 or more mm, about 11 or more mm, about 12or more mm, about 13 mm or more, about 15 mm or more, about 16 mm ormore, about 17 mm or more about 18 mm or more, about 19 mm or more, orabout 20 mm or more. In some cases, the one or more light guides has adiameter of about 16 or more mm. In some cases, the one or more lightguides has a radius of about 8.25 mm. In some cases, the one or morelight guides has a diameter of about 7 mm. In some cases, the one ormore light guides has a diameter of about 16.5 mm.

In some cases, the one or more light guides has a thickness ranging from0.5 cm to about 5 cm. In some cases, the one or more light guides has athickness of about 0.5 cm, about 1 cm, about 1.5 cm, about 2 cm, about2.5 cm, about 3 cm, about 3.5 cm, about 4 cm, about 4.5 cm, or about 5cm. In some cases, the one or more light guides has a thickness rangingfrom 1 cm to 1.5 cm. In some cases, the one or more light guides has athickness of about 1.5 cm.

In some cases, the one or more light guides can be of a circular shapeand/or other shapes as required per conditions specific to its intendeduse. In some cases, the one or more light guides comprises a circularshape. In some cases, the one or more light guides comprises acylindrical shape, a circular shape, a square shape, a spherical shape,a cone-shape, a prism-shape, or a rectangular shape. In some cases, eachof the light guides is the same shape. In some cases, each of the lightguides is a different shape. The light guides are not limited to theshapes and/or sizes as described herein and can be any shape and/or sizeas required per conditions specified to its intended use.

In some cases, the one or more light guide plates and/or light guides istransparent and made from a material selected from glass, acryl,plastic, polymethylmethacrylate (PMMA), poly-lactic acid (PLA), andepoxy. In some cases, the one or more light guide plates and/or lightguides is opaque except for the region of the light guide through whichlight passes through (e.g. the center of the light guide). In somecases, the one or more light guide plates and/or light guides is onlytransparent in the center of the light guide through which light passesthrough.

In some cases, the one or more light guide plates is made from apolymer. In some cases, the polymer is acrylic or PLA. In some cases,the one or more light guides include a reflective coating.

In some cases, the one or more light guides is configured to directlyreceive only light that is generated from the one or more LEDspositioned in the one or more circular arrays of the circuit board. Insome cases, each of one or more light guides is positioned to providefor selective illumination for each of the one or more wells of thetissue culture plate.

In some cases, the first light guide plate is connected to the circuitboard (e.g. the first circuit board or the second circuit board).

In some cases, the illumination uniformity of the light beam from lightemitted by the light source is proportional to the thickness of the oneor more light guides. In some cases, increasing the thickness of thelight guide. In some cases, increasing the thickness of the light guidefrom 1 cm to 1.5 cm decreases the difference between the edge and centerintensities of the one or more wells of the one or more wells. In somecases, the one or more light guides improves the illumination uniformityby about 10% or more, 20% or more, 30% or more, 40% or more, 50% ormore, 60% or more, or 70% or more as compared to an illumination devicewithout a light guide.

In some cases, the light guide is configured to decrease the variabilityof light intensity of the light beam emitted from the light sourcebetween each of the one or more wells of the tissue culture plate.

Optical Diffusers

In some aspects, the illumination device includes one or more opticaldiffusers. In some cases, the one or more optical diffusers comprises afirst optical diffuser and a second optical diffuser. In some cases, thefirst light guide plate is positioned between the first and secondoptical diffuser. However, optical elements beside diffusers can also beused to collimate the light to make the light beam more uniform. Anon-limiting example of an optical element other than a diffuserincludes Fresnel lenses, which is a lens that takes the shape of a flatsheet and can be used instead of a diffuser to collimate the light.

In some cases, the one or more optical diffusers comprises one or more80° circular optical diffusers. Optical diffusers can be any materialthat diffuses or scatters light. In some cases, the one or more opticaldiffusers and light guides control the uniformity and intensity of thesignals being projected onto the cell or substrate. In some cases, theone or more diffusers include one or more 80° diffuser coatings (e.g.scatter coating, full width-half max). In some cases, the diffusercoating is coated on a polycarbonate material. In some cases, thepolycarbonate has a thickness of about 0.1 inches. However, the one ormore diffusers are not limited to 80° diffuser coatings and can be anyknown diffuser coating applied to different substrates.

In some cases, the first optical diffuser is positioned between thetissue culture plate and the first light guide plate. In some cases, thesecond optical diffuser is positioned between the first light guideplate and the second light guide plate. In some cases, the first opticaldiffuser and the second optical diffuser can include optical diffusersthat are the same form of each other (e.g. made from the same material,and/or have the same dimensions, etc.). In some cases, the first opticaldiffuser and the second optical diffuser can include optical diffusersthat are different forms of each other (e.g. made from differentmaterials, and/or have different dimensions, etc.)

Optical Masks

In some cases, the illumination device includes one or more opticalmasks. In some cases, the one or more optical masks are configured toselectively block a wavelength of light outside of a core region of theone or more optical masks from reaching a detector to detect the light.In some cases, the one or more optical masks is configured to block awavelength of light from reaching the illuminated sample or substrate.

In some cases, the one or more optical masks includes an aperture. Insome cases, the one or more optical masks includes one or more cut-outfeatures. In some cases, the one or more cut-out features includes apatterned cut-out feature.

In some cases, the one or more patterned cut-out features includes acore region. In some cases, the core region includes a pattern sizeranging from 10-600 μm. In some cases, the core region includes apattern size ranging from 50-500 μm. In some cases, the core regionincludes a pattern size of about 50 or more μm, 100 or more μm, 150 ormore μm, 200 or more μm, 250 or more μm, 300 or more μm, 350 or more μm,400 or more μm, 450 or more μm, 500 or more μm, 550 or more μm, or 600or more μm.

In some cases, the one or more patterned cut-out features is a slit. Insome cases, the one or more patterned cut-out features is a curved slit(e.g., an arc). In some cases, the one or more patterned cut-outfeatures is a circle, rectangle, square, or triangle. In some cases, thepatterned cut-out feature can be of a slit shape and/or other shapes asrequired per conditions specific to its intended use.

In some cases, the one or more optical masks is configured toselectively block the passage of light outside of the core region.

In some cases, the one or more optical masks are made of an opaquematerial. In some cases, the core region of the one or more opticalmasks does not include the opaque material from which the optical maskis made from (e.g. the core region includes an aperture or a patternedslit in the material). In some cases, the one or more optical masksinclude a combination of a transparent material and an opaque material.In some cases, the combination of a transparent material and an opaquematerial provides for greyscale modulation of the light pattern. In somecases, the core region of the optical mask includes a transparentmaterial, and the material outside of the core region is an opaquematerial.

In some cases, the one or more optical masks is adhered to the one ormore wells of the tissue culture plate. In some cases, the one or moreoptical masks is positioned on a surface of one or more wells of thetissue culture plate. In some cases, the one or more optical masks ispositioned on a bottom surface of the one or more wells. In some cases,the one or more optical masks is positioned on a bottom outer surface ofthe one or more wells. In some cases, the core region of the one or moreoptical masks is positioned in the center of an outer surface of the oneor more wells. In some cases, the one or more optical masks ispositioned on the one or more wells of the tissue culture plate. In somecases, the one or more optical masks is positioned on an outer surfaceof the coverglass bottom. In some cases, the one or more optical masksis positioned at a distal end (e.g. the closed-ended outer surface ofthe one or more wells) of the one or more wells of the tissue cultureplate relative to an open ended surface of the one or more wells. Insome cases, the one or more optical masks is positioned on a surface ofone or more wells of the tissue culture plate.

In some cases, the one or more optical masks includes an optical maskpositioned on each of the one or more wells of the tissue culture plate.In some cases, the one or more optical masks comprises 24 optical masks,each mask positioned on each of the 24 wells of the tissue cultureplate. In some cases, the one or more optical masks includes an opticalmask positioned on each of the one or more wells of the tissue cultureplate. In some cases, the one or more optical masks comprises 96 opticalmasks, each mask positioned on each of the 96 wells of the tissueculture plate. In some cases, the one or more optical masks includes anoptical mask positioned on each of the one or more wells of the tissueculture plate. In some cases, the one or more optical masks comprises384 optical masks, each mask positioned on each of the 384 wells of thetissue culture plate.

In some cases, the optical mask includes a photo mask or an intensitymask. An optical mask can include a material, coating, and/or plate withholes or transparencies that allow light to pass through in a definedpattern. In some cases, the optical mask absorbs light to varyingdegrees and can be patterned as required per conditions specific to itsintended use. In some cases, the optical mask is an intensity mask. Insome cases, the intensity mask is fully absorbing (e.g. opaque, dark),or not absorbing (e.g. transparent, bright), or a combination thereof.In some cases, the intensity mask is made from adhesive vinyl. In somecases, the adhesive vinyl is polyvinyl chloride (PVC). In some cases,the intensity mask is made from Biaxially Oriented Polypropylene.

In some cases, the optical mask is a phase mask. In some cases, thephase mask is a phase shift mask. A phase mask is used to modulate thephase of light in order to change the light intensity. The phasemodulation results in constructive and destructive interference thatgenerates a pattern of light intensity, which can be similar to a lightpattern generated with an intensity mask. It can be used in combinationwith an intensity mask, which is then called a phase-shift mask. In somecases, the phase mask is made from glass (e.g. quartz).

In some cases, the illumination pattern when the light beam contacts theone or more wells includes a 0.5 mm diameter of light, 1.0 mm diameterof light, 1.5 mm diameter of light, or a 2 mm diameter of light emittedto the one or more wells. In some cases, the illumination patternincludes a 1.5 mm diameter of light emitted to the one or more wells.

Detector

In some aspects, the illumination device includes one or more detectors.In some aspects, the illumination device can be used in combination witha microscope, a spectrophotometer, a detector, or a robotic handler.Non-limiting examples of microscopes include a fluorescence microscope,a confocal laser scanning microscope, and/or a bright-field microscope),a spectrophotometer, or a detector. In some cases, the detector is aphotomultiplier tube, a charged coupled device (CCD), or a complementarymetal oxide semiconductor (CMOS) sensor. In some cases, the detectors ofinterest are configured to measure collected light at one or morewavelengths, such as at 2 or more wavelengths, such as at 5 or moredifferent wavelengths, such as at 10 or more different wavelengths, suchas at 25 or more different wavelengths, such as at 50 or more differentwavelengths, such as at 100 or more different wavelengths, such as at200 or more different wavelengths, such as at 300 or more differentwavelengths and including measuring light emitted by a sample in theflow stream at 400 or more different wavelengths. In some embodiments,detectors are configured to measure collected light over a range ofwavelengths (e.g., 200 nm to 1000 nm). In some embodiments, detectorsare configured to collect spectra of light over a range of wavelengths.In some embodiments, an optical imaging system may include one or moredetectors configured to collect spectra of light over one or more of thewavelength ranges of 200 nm to 1000 nm. In some embodiments, detectorsare configured to measure light emitted by a cell or substrate in theone or more wells of a tissue culture plate at one or more specificwavelengths. For example, the one or more detectors are configured tomeasure light at one or more of 350 nm, 370 nm, 400 nm, 410 nm, 450 nm,518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm,667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm,617 nm and any combinations thereof. In some embodiments, one or moredetectors may be configured to be paired with specific fluorophores,such as those used in a fluorescence assay.

In some cases, the one or more detectors is positioned about 5 or moremm away from the light source. In some cases, the one or more detectorsis positioned about 10 mm or more, 15 mm or more, 20 mm or more, or 25mm or more away from the light source. In some cases, the one or moredetectors is positioned 21 mm away from the light source.

Graphical User Interface (GUI)

In some cases, the illumination device can be controlled with agraphical user interface (GUI) to communicate wirelessly with thecontroller of the illumination device. In some cases, the GUI can beused to program the illumination parameters of the one or more wells. Insome cases, the GUI can be used to wirelessly program the illuminationintensity and temporal (e.g. time) sequences of each of the one or morewells. In some cases, the GUI provides for a user to input a desiredillumination parameter for each of the one or more wells. In some cases,the GUI provides for wireless upload of the illumination parameters tothe controller (e.g. the microcontroller and/or the LED driver) on theillumination device.

In some cases, the GUI allows the user to set each channel of the LEDdriver (e.g. in a 24 channel LED driver or a 96-channel LED driver) withone or more illumination parameters. In some cases, the GUI allows theuser to set each channel to, for example, constant illumination at aspecified intensity; blinking illumination at a specified intensity,duty cycle, and time period; and a series of linear or sinusoidalfunctions at specified illumination parameters.

In some cases, the GUI provides for multiple piecewise functions thatcan be programmed in a time sequence.

Illumination Parameters

In some cases, the illumination device can spatially and temporallycontrol illumination of a cell or a substrate with one or moreillumination patterns.

In some cases, the illumination parameters can include, but are notlimited to, frequency of light emitted from the light source, dutycycle, duration of light emitted from the light source, and specificpatterns of illumination (e.g. pulsed illumination). In some cases, theone or more illumination parameters is selected from an illuminationintensity, an illumination duration, an illumination pattern, and acombination thereof.

In some cases, the illumination intensity ranges from about 0.005 μW/mm²to about 20 μW/mm². In some cases, the illumination intensity rangesfrom about 0.005 μW/mm² to about 10 μW/mm². In some cases, theillumination intensity ranges from about 0.005 μW/mm² to about 20mW/mm².

In some cases, the illumination pattern is a pulsing pattern, wherelight is pulsed in a millisecond time frame. In some cases, anillumination duration ranges from about 0.5 or more ms, 1 or more ms, 2or more ms, 3 or more ms, 4 or more ms, 5 or more ms, 6 or more ms, 7 ormore ms, 8 or more ms, or 10 or more ms. In some cases, the pulsedillumination includes a pulse duration of 1 or more ms.

In some cases, the illumination pattern is a pulsing pattern, wherelight is pulsed in a millisecond time frame. In some cases, theillumination device further includes a pulse generator configured topulse the light emitted from the light source.

In some cases, the illumination pattern includes a sinusoidal or linearpattern with a pulsing frequency of 1 or more Hz.

In some cases, the illumination pattern includes a blinking pattern witha pulsing frequency of about 1 or more Hz, 10 or more Hz, 20 or more Hz,30 or more Hz, 40 or more Hz, 50 or more Hz, 60 or more Hz, 70 or moreHz, 80 or more Hz, 90 or more Hz, 100 or more Hz, 100 or more Hz, 110 ormore Hz, 120 or more Hz, 130 or more Hz, 140 or more Hz, or 150 or moreHz. In some cases, the illumination pattern includes a blinking patternwith a pulsing frequency of about 100 Hz.

In some cases, the illumination pattern includes a 0.5 mm diameter oflight, 1.0 mm diameter of light, 1.5 mm diameter of light, or a 2 mmdiameter of light emitted to the one or more wells. In some cases, theillumination pattern includes a 1.5 mm diameter of light emitted to theone or more wells (e.g. the diameter of the light beam when it contactsthe one or more wells).

In some cases, the illumination duration includes illumination of theone or more wells for 1 or more hours. In some cases, the illuminationduration includes illumination of the one or more wells for 1 or moreweeks. In some cases, the illumination duration ranges from about 0.5 ormore ms, 1 or more ms, 2 or more ms, 3 or more ms, 4 or more ms, 5 ormore ms, 6 or more ms, 7 or more ms, 8 or more ms, or 10 or more ms. Insome cases, the illumination duration ranges from about 0.5 or more s, 1or more s, 2 or more s, 3 or more s, 4 or more s, 5 or more s, 6 or mores, 7 or more s, 8 or more s, or 10 or more s. In some cases, theillumination duration ranges from about 1 or more minutes, 2 or moreminutes, 3 or more minutes, 4 or more minutes, 5 or more minutes, 6 ormore minutes, 7 or more minutes, 8 or more minutes, or 10 or moreminutes.

In some cases, the one or more wells can be illuminated with red, amber,yellow, green, blue, or white light. In some cases, the GUI can set thecolor of light corresponding to a specific wavelength.

In some cases, the GUI allow the user to set a wavelength of light forwhich light can be emitted. In some cases, the light can have awavelength ranging from 200 to 1000 nm. In some cases, the light canhave a wavelength ranging from about 350 to about 410 nm. In some cases,the light can have a wavelength of about 470 nm and about 510 nm or canhave a wavelength of about 490 nm. In some cases, the light can have awavelength of about 470 nm. In some cases, the light can have awavelength of about 445 nm. In some cases, the light can have awavelength ranging from about 530 to about 595 nm. In some cases, thelight can have a wavelength of about 530 nm. In some cases, the lightcan have a wavelength of about 560 nm. In some cases, the light can havea wavelength of about 542 nm. In some cases, the light can have awavelength of about 546 nm. In some cases, the light can have awavelength ranging from about 580 and 630 nm. In some cases, the lightcan be at a wavelength of about 589 nm or the light can have awavelength greater than about 630 nm (e.g. less than about 740 nm). Inanother embodiment, the light has a wavelength of around 630 nm.

A device of the present disclosure can be part of a system that providesfor spatial and temporal control of light using the illumination device.For example, in some cases, a system of the present disclosure includes:an illumination device of the present disclosure; a tissue culture plateincluding one or more wells; and one or more of: i) a microscope; ii) aspectrophotometer; iii) a detector; iv) a power source; v) a coolingfan; vi) a heat sink; vii) a graphical user interface; and viii)computer hardware and software for controlling the illumination device.

Methods

The present disclosure provides methods for spatially and temporallycontrolling light using the system and/or illumination devices of thepresent disclosure. According to aspects of the present disclosure, themethod includes activating a cell or a substrate with light, wherein thecell or the substrate is within one or more wells of a tissue cultureplate, wherein the light is generated by a system comprising: a lightsource operably adjacent to a circuit board and configured to producelight; one or more light guide plates comprising one or more lightguides; one or more optical masks positioned on a surface of the one ormore wells of the tissue culture plate; a controller; a computerreadable medium, comprising instructions that, when executed by thecontroller, cause the controller to: illuminate the cell or thesubstrate in the one or more wells with the light from the light source;and spatially and temporally control illumination of the cell or thesubstrate in the one or more wells with one or more illuminationparameters, wherein the one or more light guides is configured toprovide uniform illumination of the light in the one or more wells ofthe tissue culture plate.

Tissue Culture Plate

In some aspects, method includes stimulating and/or activating a cell ora substrate in a tissue culture plate that is positioned adjacent to theillumination device. In some cases, the culture plate is a tissueculture plate (e.g. a cell culture plate or a multi-well plate). In somecases, the illumination device is reversibly connected to a tissueculture plate. By “adjacent”, as used herein in its conventional senseto refer to be in contact with, connected, linked, fastened, orpositioned on a surface of the illumination device. In some cases, anillumination device positioned adjacent to a culture plate includes agap or space between the tissue culture plate and the illuminationdevice.

In some cases, the tissue culture plate includes one or more wells. Insome cases, the tissue culture plate includes 24 wells. In some cases,the tissue culture plate includes 96 wells. In some cases, the tissueculture plate includes 384 wells.

In some cases, the tissue culture plate is made from an opaque polymer.In some cases, the tissue culture plate is made from a black polymer. Insome cases, the tissue culture plate is made from a material thatprevents light from bleeding through between the one or more wells.

In some cases, the tissue culture plate includes a coverglass bottom. Insome cases, the one or more wells include a coverglass bottom. In somecases, the coverglass bottom has a thickness ranging from 150-200 μm. Insome cases, the coverglass bottom has a thickness of about 150 μm, 160μm, 170 μm, 180 μm, 190 μm, or 200 μm.

In some cases, the method includes placing the illumination deviceconnected to the tissue culture plate in an incubator. In some cases,the method includes controlling the illumination device connected to thetissue culture plate placed in an incubator. In some cases, controllingthe illumination device connected to the tissue culture plate includescontrolling the illumination device wirelessly removing the illuminationdevice connected to the tissue culture plate from the incubator.

Cells

In some cases, the tissue culture plate includes one or more wells. Insome cases, the method includes placing (e.g. administering a cell viapipette, automated robotic grip, or any other means of administering) acell in the one or more wells (e.g. one or more cells). In some cases,the method includes aspirating and/or replacing a fluid (e.g. cellculture media, a buffer, or any other solution within the one or morewells) from the one or more wells. In some cases, the cell is amammalian cell, a bacterial cell, a yeast cell, or a plant cell.Non-limiting examples of mammalian cells include a stem cell, aprogenitor cell, a neural cell, or a cardiac cell. In some cases, thecell is a stem cell or a progenitor cell. In some cases, the cell is abacterial cell. In some cases, the cell is a green algeo. In some cases,the cell is a cyanobacteria. In some cases, the cell is spirulina, orSynechococcus elongatus. Non-limiting examples of bacterial cellsinclude Bacillus subtilis, Escherichia coli, Streptomyces and Salmonellatyphimuium cells. In some cases, the cell is a yeast cell. Anon-limiting example of a yeast cell is a yeast cell of the speciesSaccharomyces cerevisiae. In some cases, the cell is a plant cell.

In some cases, the method includes spatially and temporally controllingcell signaling and differentiation.

In some cases, the method further includes screening for phototoxicityof the cell in response to light. In some cases, the method furtherincludes screening for a candidate agent to determine whether thecandidate agent modulates an activity of the cell (e.g. activation,deactivation, signaling, differentiation).

In some cases, the method includes expressing a protein in the cell. Insome cases, the protein is a light activated protein. In some cases, themethod includes stimulating the light activated protein. In some cases,the method includes stimulating the light activated protein expressed inthe cell.

In some cases, the protein is a fluorescent protein. In some cases, themethod further comprises screening for a fluorescent sensor expressed inthe cell. In some cases, the fluorescent protein is a yellow fluorescentprotein, a red fluorescent protein, a green fluorescent protein, or acyan fluorescent protein. In some cases, the method further includesscreening for the fluorescent protein expressed in the cell in responseto light. In some cases, the cell expresses a genetically encodedfluorescent sensor derived from a fluorescent protein. In some cases,the method further includes screening for the genetically encodedfluorescent sensor expressed in the cell.

Light-Activated Proteins

In some cases, the method includes expressing a light-activated protein(e.g. in the cell). In some cases, the method includes stimulating thelight activated protein. In some cases, stimulating the light-activatedprotein activates the light-activated protein in response to light fromthe light source. In some cases, stimulating the light-activated proteinactivates the cell expressing the light-activated protein in response tolight from the light source. In some cases, the method includes fusingthe light activated protein to a c-terminal domain of a lipoproteinreceptor-related protein 6 (LRP6).

In some aspects, the light activated protein induces proteininteractions. For example, light activated proteins and portions thereofcan change conformation upon light absorption, for example, usingproteins such as rhodopsins, phytochromes, and cryptochromes, and LOVdomains from phototropins and FKF1 (Airan et al. (2009) Nature458:1025-1029; Inoue et al. (2005) Nat. Methods 2:415-418; Kennedy etal. (2010) Nat. Methods 7:973-975; Levskaya et al. (2009) Nature461:997-1001; Szobota et al. (2007) Neuron 54:535-545; Wu et al. (2009)Nature 461:104-108; and Yazawa et al. (2009) Nat. Biotechnol.27:941-945).

In some cases, the light activated protein is selected from acryptochrome, which is a blue light-sensitive flavoprotein found inplants, animals and microbes; a photoactive yellow protein (PYP)photosensor, which is found in certain bacteria; a photoreceptor ofblue-light using flavin adenine dinucleotide (BLUF) and Light, Oxygen,or Voltage sensing (LOV) types, which are plant and bacterialphotoreceptors; and a phytochrome, which is used by plants and microbesand are sensitive to light in the red-to-NIR region.

In some cases, the light activated protein is a light-inducibledimerizer. In some cases, the dimerizer is the CRY2/CIB system, based ona light-dependent interaction between Arabidopsis cryptochrome 2(AtCRY2) and an interacting partner, CIB 1. In some cases, the dimerizeris the Phy/Pif system. In some cases, the dimerizer is the BphP1/PpsR2system.

In some cases, the light activated protein is a caged protein domain. Insome cases, the caging domain is a LOV domain isolated from the plantphotosensor phototropin 1 (phot1). In some cases, the LOV domain isLOV2. In some cases, the light activated protein is a phytochrome. Insome cases, the phytochrome contains a LOV domain, such as phototropin1, white collar-1 (WC-1), white collar-2 (WC-2), photoactive yellowprotein (PYP), Phy3, and VVD. In some cases, the phytochrome isphytochrome B (phyB). PhyB binds to a class of target transcriptionfactors termed phytochrome-interacting factors (Pifs). Thislight-induced, reversible Phy/Pif dimerization is harnessed tooptogenetically stimulate protein-protein interactions in mammaliancells. In some cases, the phytochrome is a bacterial bathyphytochromeBphP1 that interacts with its binding partner PpsR2.

In some cases, the light activated protein is a reactive oxygen species.In some cases, the light activated protein is a genetically encodedROS-generating protein. In some cases, the light activated protein is amini singlet oxygen generator (miniSOG), which is a 106 amino acid greenfluorescent flavoprotein generated from Arabidopsis phototropin 2. Insome cases, the light activated protein is KillerRed. KillerRed is aphototoxic fluorescent protein derived from a homolog of GFP, anm2CP.

In some cases, the light activated protein is a photoreceptor UVR8 (UVResistance Locus 8) has been identified and characterized as a distinctplant photoreceptor that perceives light signals in the UV-B regionusing intrinsic Trp residues as chromophores

In some cases, the light activated protein is CarH, a bacterialtranscriptional regulator that controls the biosynthesis of carotenoidsin response to light.

In some aspects, stimulating and/or activating the light activatedproteins causes membrane depolarization of cell. In some cases, thelight activated protein can include depolarizing light-activatedproteins. Non-limiting examples of depolarizing light-activated proteinsinclude, e.g., members of the Channelrhodopsin family of light activatedprotein proteins such as Chlamydomonas rheinhardtii channelrhodopsin 2(ChR2); a step-function opsin (SFO); a stabilized SFO (SSFO); a chimericopsin such as C1V1; a Volvox carteri-derived channelrhodopsin (VChR1),etc. Such light-responsive polypeptides can be used to promote neuralcell membrane depolarization in response to a light stimulus.

In some aspects, the method includes deriving the light activatedprotein from Chlamydomonas reinhardtii, wherein the cation channelprotein can be capable of transporting cations across a cell membranewhen the cell is illuminated with light.

In some cases, the method includes activating the light activatedprotein with a wavelength between about 460 and about 495 nm or can havea wavelength of about 480 nm. In some cases, activating the lightactivated protein includes pulsing the light having a temporal frequencyof about 100 Hz to activate the light-responsive protein. In someembodiments, activating the light activated protein by pulsing the lighthaving a temporal frequency of about 100 Hz can cause depolarization ofthe neurons expressing the light activated protein. The light activatedprotein can additionally comprise substitutions, deletions, and/orinsertions introduced into a native amino acid sequence to increase ordecrease sensitivity to light, increase or decrease sensitivity toparticular wavelengths of light, and/or increase or decrease the abilityof the light activated protein to regulate the polarization state of theplasma membrane of the cell. Additionally, the light activated proteincan contain one or more conservative amino acid substitutions and/or oneor more non-conservative amino acid substitutions. The light-responsiveproton pump protein comprising substitutions, deletions, and/orinsertions introduced into the native amino acid sequence suitablyretains the ability to transport cations across a cell membrane.

In some aspects, stimulating and/or activating the light activatedproteins causes membrane hyperpolarization of cell. In some cases, thelight-activated protein is a hyperpolarizing light-activated protein.Non-limiting examples of suitable light-responsive polypeptides to beexpressed in a cell include, e.g., the Halorhodopsin family oflight-responsive chloride pumps (e.g., NpHR, NpHR2.0, NpHR3.0, NpHR3.1).As another example, the GtR3 proton pump can be used to promote cellmembrane hyperpolarization in response to light. As another example,eArch (a proton pump) can be used to promote neural cell membranehyperpolarization in response to light. As another example, an ArchTopsin protein or a Mac opsin protein can be used to promote neural cellmembrane hyperpolarization in response to light.

In some cases, the cell in the one or more wells of the tissue cultureplate is a stem cell or a progenitor cell. In some cases, methodincludes expressing a light activated protein in the cell. In somecases, the method includes inducing Wnt/β-catenin signaling in the stemcell in response to light. In some cases, stimulating and/or activatingthe stem cell expressing the light-activated protein inducesWnt/β-catenin signaling in the stem cell in response to light. In somecases, the method includes fusing the light activated protein to ac-terminal domain of a lipoprotein receptor-related protein 6 (LRP6). Insome cases, the method includes inducing differentiation of the stemcell into a mesenchymal stem cell via activation of the Wnt/β-cateninsignaling. In some cases, activating and/or stimulating the stem cell,in response to light, expresses a brachyury (Bra) protein.

Substrates

Aspects of the present disclosure include stimulating and/or activatinga substrate in one or more wells of the tissue culture plate. In somecases, the method includes polymerizing a substrate in response tolight. In some cases, the method includes photopolymerizing thesubstrate in response to light. In some cases, the stimulating and/oractivating the substrate photopolymerizes the substrate in response tolight (e.g. light-based activatable signal). In some cases, the methodincludes spatially and temporally controlling the light as described inthe present disclosure, wherein controlling the light provides forspatial and temporal control of photopolymerization of the substrate inresponse to the light.

In some cases, the method includes photopatterning of the substrate inresponse to light. In some cases, stimulating and/or activating thesubstrate provides for photopatterning of the substrate in response tolight.

In some cases, the substrate is a polymer. In some cases, polymer is atleast one of polylactic acid, poly(lactic-co-glycolic) acid,poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyrate,polyhydroxyalcanoic acid, chitosan, hyaluronic acid (HA), a hydrogels,poly(2-hydroxyethyl-methacrylate), poly(ethylene glycol),poly(L-lactide) (PLA), poly(dimethysiloxane) (PDMS),poly(methylmethacrylate) (PMMA), poly(glycerol sebacate),poly(octamethylene maleate (anhydride) citrate) (POMaC), POMaC withoutcitric acid, poly(.epsilon.-caprolactone), polyurethane, silk, ananofabricated material, a co-polymer, a blended polymer, or acombination thereof.

In some cases, the polymer includes water-soluble polymer chains. Insome cases, the polymer includes water-soluble polymer chains with across-linker group. In some cases, the method includes crosslinkingpolymer chains of the substrate in response to light. In some cases, thepolymer chains include one or more methacrylate groups. In some cases,the method includes photopolymerizing the one or more methacrylategroups in response to the light. Methacrylated HA is widely used asscaffolds of extracellular matrix mimicking biomaterials, while themethacrylate groups can self-crosslink in the presence of aphotoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).LAP has a broad adsorption band ranging from 350 nm to 410 nm. Onemolecule of LAP decomposes and dissociates into two radicals followingphoton adsorption, which further triggers the Michael Addition-typereaction between two methacrylate groups and thus forms crosslinkswithin the polymer chains. In some cases, the method includesadministering (e.g. introducing, placing, applying, pipetting) aphotoinitiator on the polymer.

In some cases, the substrate is a polymer. In some cases, the polymer isa hydrogel. In some cases, the substrate is a hydrogel. In some cases,the method includes crosslinking polymers within a hydrogel. In somecases, the substrate is a hydrogel made from hyaluronic acid. In somecases, the hydrogel includes an adhesion motif (protein orprotein-derived peptide ligands), an antibody, a growth factors, and/ora gene-encoding nucleic acid, or other bioactive molecules to promotebiocompatibility of the hydrogel. In some cases, the method includesintroducing one or more of an adhesion motif, an antibody, a growthfactors, and/or a gene-encoding nucleic acid, and other bioactivemolecules into the hydrogel. In some cases, the hydrogel includesadhesion motifs, such as RGD peptides. In some cases, stimulating and/oractivating the hydrogel provides for photopatterning of the hydrogel(e.g. stimulating and/or activating at a wavelength in the ultravioletspectrum). In some cases, the method includes photopatterning thehydrogel with one or more an adhesion motif, an antibody, a growthfactors, and/or a gene-encoding nucleic acid, or other bioactivemolecules. In some cases, stimulating and/or activating the hydrogelprovides for photopatterning of the hydrogel with one or more anadhesion motif, an antibody, a growth factors, and/or a gene-encodingnucleic acid, or other bioactive molecules.

Other non-limiting examples of hydrogels include hydrogels made frompolyvinyl alcohol, sodium polyacrylate, an acrylate polymer, agarose,methylcellulose, or hyaluronan.

In some cases, the polymer includes one or more methacrylate groups. Insome cases, the method includes introducing a photoinitiator to thepolymer. In some cases, the photoinitiator is lithiumphenyl-2,4,6-trimethylbenzoylphosphinate (LAP). In some cases, themethod includes photopatterning the polymer in response to light. Insome cases, stimulating and/or activating the hydrogel provides forphotopatterning of the polymer in response to light. In some cases, theactivating provides for crosslinkage of a first methacrylate group and asecond methacrylate group within the polymer. In some cases, the methodincludes crosslinking a first methacrylate group and a secondmethacrylate group within the polymer. In some cases, the reaction ratebetween the one or more methacrylate groups is proportional to lightintensity. In some cases, the number of crosslinks formed by the one ormore methacrylate groups is proportional to light intensity. In somecases, the method includes modulating the stiffness of the polymer inresponse to light. In some cases, the stiffness of the polymer isproportional to the light intensity and/or the duration of lightexposure of the light emitted by the light source.

In some cases, the hydrogel includes one or more cells. In some cases,the method further includes screening for interactions of the one ormore cell with an extracellular matrix within the hydrogel.

Light Source

In some cases, the method includes illuminating the one or more wells ofthe illumination device using a light source. In some cases, the methodincludes producing or generating uniform light with the light source. Insome cases, the method includes positioning the light source adjacent toa circuit board. In some cases, the method includes connecting lightsource to a circuit board. In some cases, the method includes operablyconnecting the light source to a circuit board.

In some cases, the light source can be a light emitting diode (LED). Insome cases, the light source includes one or more LEDs. In some cases,the LED can generate white, blue, red, and/or green light. In somecases, the LED can generate amber and/or yellow light. In some cases,the LEDs are micro LEDs. In some cases, the LEDs are embedded into acircular array of the circuit board. In some embodiments, the lightsource is a solid state laser diode or any other means capable ofgenerating light. The light generating means can generate light havingan intensity sufficient to activate a cell, protein, and/or a substrate.In some cases, the light includes an irradiance (e.g. light afterpassing through the optics) having an intensity of any of about 0.005μW/mm², 0.006 μW/mm², 0.0007 μW/mm², 0.008 μW/mm², 0.009 μW/mm², 0.01μW/mm², 0.02 μW/mm², 0.03 μW/mm², 0.04 μW/mm², 0.05 μW/mm², 0.1 μW/mm²,0.2 μW/mm², 0.3 μW/mm², 0.4 μW/mm², 0.5 μW/mm², about 0.6 μW/mm², about0.7 μW/mm², about 0.8 μW/mm², about 0.9 μW/mm², about 1.0 μW/mm², about1.1 μW/mm², about 1.2 μW/mm², about 1.3 μW/mm², about 1.4 μW/mm², about1.5 μW/mm², about 1.6 μW/mm², about 1.7 μW/mm², about 1.8 μW/mm², about1.9 μW/mm², about 2.0 μW/mm², about 2.1 μW/mm², about 2.2 μW/mm², about2.3 μW/mm², about 2.4 μW/mm², about 2.5 μW/mm², about 3 μW/mm², about3.5 μW/mm², about 4 μW/mm², about 4.5 μW/mm², about 5 μW/mm², about 5.5μW/mm², about 6 μW/mm², about 7 μW/mm², about 8 μW/mm², about 9 μW/mm²,about 10 μW/mm², about 11 μW/mm², about 12 μW/mm², about 13 μW/mm²,about 14 μW/mm², about 15 μW/mm², about 16 μW/mm², about 17 μW/mm²,about 18 μW/mm², about 19 μW/mm², about 20 μW/mm², about 21 μW/mm²,about 22 μW/mm², about 23 μW/mm², about 24 μW/mm², or about 25 μW/mm²,inclusive, including values in between these numbers. In some cases, thelight includes an irradiance having an intensity ranging from about0.0001 to about 25 μW/mm², about 25 to 50 μW/mm², about 50-100 μW/mm²,about 100-150 μW/mm², or 150-200 μW/mm². In other embodiments, thelight-generating means produces light having a frequency of at leastabout 100 Hz. In some cases, the light source produces light having anintensity of any of about 0.05 mW/mm², 0.1 mW/mm², 0.2 mW/mm², 0.3mW/mm², 0.4 mW/mm², 0.5 mW/mm², about 0.6 mW/mm², about 0.7 mW/mm²,about 0.8 mW/mm², about 0.9 mW/mm², about 1.0 mW/mm², about 1.1 mW/mm²,about 1.2 mW/mm², about 1.3 mW/mm², about 1.4 mW/mm², about 1.5 mW/mm²,about 1.6 mW/mm², about 1.7 mW/mm², about 1.8 mW/mm², about 1.9 mW/mm²,about 2.0 mW/mm², about 2.1 mW/mm², about 2.2 mW/mm², about 2.3 mW/mm²,about 2.4 mW/mm², about 2.5 mW/mm², about 3 mW/mm², about 3.5 mW/mm²,about 4 mW/mm², about 4.5 mW/mm², about 5 mW/mm², about 5.5 mW/mm²,about 6 mW/mm², about 7 mW/mm², about 8 mW/mm², about 9 mW/mm², about 10mW/mm², about 11 mW/mm², about 12 mW/mm², about 13 mW/mm², about 14mW/mm², about 15 mW/mm², about 16 mW/mm², about 17 mW/mm², about 18mW/mm², about 19 mW/mm², about 20 mW/mm², about 21 mW/mm², about 22mW/mm², about 23 mW/mm², about 24 mW/mm², or about 25 mW/mm², inclusive,including values in between these numbers.

In some aspects, the method includes externally activating the lightsource by a controller. In some cases, the controller includes aprocessor. In some cases, the method includes supplying power to thecontroller via a power source. In some cases, the method includesmounting a power source to a transmitting coil. In some cases, themethod includes, the method includes connecting a battery to the powersource, for providing power thereto (e.g. to the controller). In somecases, the method includes connecting a switch to the power source,allowing an individual to manually activate or deactivate the powersource.

In some cases, the method includes independently illuminating each ofthe one or more wells of the tissue culture plate. In some cases, themethod includes illuminating each of the one or more wells of the tissueculture plate by the one or more LEDs.

Circuit Board

In some aspects, the illumination device includes one or more circuitboards. In some cases, the method includes positioning a light sourceadjacent to the circuit board. In some cases, the method includesconnecting a light source to the circuit board. In some cases, the lightsource includes one or more LEDs. In some cases, the method includesgenerating a printed circuit board (PCB). In some cases, theillumination device includes one or more circuit boards. In some cases,the circuit board includes one or more circular arrays. In some cases,the illumination device includes a first circuit board (PCB1). In somecases, the PCB1 includes electronics for LED control. In some cases, theillumination device further comprises a power distribution board. Insome cases, the illumination device includes a second circuit board(PCB2). In some cases, the PCB2 is a power distribution board. In somecases, the PCB1 contains solder pads for a circular array of 5 LEDs inorder to emit light from the 5 LEDs to one well of the tissue cultureplate (e.g. a 24 well tissue culture plate). In some cases, the 5 LEDsare connected to the circular array of the circuit board in series.

In some cases, the one or more LEDs are symmetrically and radiallydistributed on one or more circular arrays on the circuit board. In somecases, each of the one or more circular arrays has a radius ranging fromabout 2-10 mm (e.g. 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or10 mm). In some cases, each of the one or more circular arrays has aradius of about 5 mm.

In some cases, the method includes distributing 5 LEDs symmetrically andradially on each of the one or more circular arrays of the circuitboard. In some cases, the circuit board includes 24 circular arrays. Insome cases, the method includes positioning one or more circular arraysbelow the one or more wells of the tissue culture plate. In some cases,the method includes illuminating one well of the one or more wells inthe tissue culture plate with 5 LEDs on the circular array.

In some cases, the method includes distributing 1 LED symmetrically andradially on each of the one or more circular arrays of the circuitboard. In some cases, the circuit board includes 96 circular arrays. Insome cases, the method includes positioning 1 LED at approximately thecenter of each circular array.

In some cases, the method includes positioning one or more LEDs on thecircuit board below the one or more wells of the tissue culture plate.

In some aspects, the method includes reducing heat and/or moving heataway from the illumination device and/or tissue culture plate through aheat sink mounted on the circuit board. In some cases, the methodincludes mounting a heat sink on the circuit board with a thermallyconductive adhesive. In some cases, the method includes mounting a heatsink onto the bottom surface of the first circuit board (e.g. PCB1). Insome cases, the method includes mounting a heat sink using a thermallyconductive adhesive. In some cases, the thermally conductive adhesive isArtic Silver, ASTA-7G. In some cases, the method includes mounting theheat sink onto the circuit board (e.g. first circuit board) in a regionwithout silk screen and thermally conductive electrical vias that drawheat away from the one or more LEDs.

In some cases, the method includes cooling the illumination deviceand/or tissue culture plate. In some cases, the method includes coolingthe illumination device and/or tissue culture plate with one or morecooling fans. In some cases, the illumination device includes twocooling fans. In some cases, the illumination device includes threecooling fans. In some cases, the method includes positioning one or morecooling fans on the outer edges of the circuit board. In some cases, themethod includes electrically connecting the first circuit board to theone or more cooling fans.

In some aspects, the method includes connecting the illumination deviceto a power supply. In some cases, the method includes operablyconnecting the illumination device to the power supply. In some cases,the method includes electrically connecting the illumination device tothe power supply. In some cases, the method includes connecting thepower supply to the second circuit board (e.g. PCB2) of the illuminationdevice. In some cases, method includes supplying power to the one ormore cooling fans and the controller through switching voltageregulators.

Controller

In some aspects, the method includes controlling the illumination devicewith a controller. In some cases, controlling the illumination devicewith a controller provides for independently illuminating each of theone or more wells of the tissue culture plate.

In some cases, the controller is a microcontroller. In some cases, thecontroller is a Raspberry Pi microcontroller. In some cases, the methodincludes mounting a tissue culture plate in a position on theillumination device such that the method provides for illuminating thetissue culture plate from the bottom.

In some cases, the method includes controlling the illumination devicewith a controller, wherein the controller includes an LED driver. Insome cases, the method includes controlling the illumination device withan LED driver. In some cases, the LED driver includes one or morechannels. In some cases, the LED driver is a 24-channel LED driver. Insome cases, the LED driver is a 96-channel LED driver. In some cases,the LED driver is a 384-channel LED driver. In some cases, the methodincludes controlling two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, or ten ormore LED drivers. In some cases, the illumination device includes fourLED drivers. In some cases, the method includes controlling,independently, of each of the channels of the LED driver (e.g. a24-channel LED driver, a 96 channel LED driver, or a 384-channel LEDdriver). In some cases, the method includes independently illuminatingeach of the one or more wells of the tissue culture plate with thecontroller.

In some cases, the method includes connecting the illumination device toa power supply. In some cases, the method includes connecting the powersupply to the LED driver of the illumination device. In some cases, themethod includes connecting the power supply to the second circuit boardthrough a barrel power jack to power the one or more LEDs through theLED driver. In some cases, the method includes powering the secondcircuit board to power the one or more LEDs through the LED driver.

In some cases, the method includes electrically connecting the LEDdriver to the circuit board. In some cases, the method includes mountingthe LED driver and electrically connecting the LED driver to the firstcircuit board of the illumination device. In some cases, the methodincludes mounting the microcontroller and electrically connecting themicrocontroller to the first circuit board. In some cases, the methodincludes mounting the second circuit board (e.g. PCB2) and electricallyconnecting the first circuit board to the first circuit board (PCB1).

In some cases, the method includes controlling the one or more wells ofthe tissue culture plate with one or more channels of the LED driver. Insome cases, the LED driver is a 24-channel LED driver. In some cases,the method includes controlling one or more LEDs with the LED driver. Ina non-limiting example, for 24-well illumination (e.g. illumination of24 wells in a 24 tissue culture plate), a first circuit board includes24 circular arrays containing 5 LEDs radially and symmetricallydistributed on each of the 24 circular arrays. In some cases, the methodincludes independently controlling illumination of each well in the 24tissue culture plate with the 24-channel LED driver, wherein eachchannel of the LED driver controls one well in the 24 well plate.Another non-limiting example includes 96-well illumination (e.g.illumination of 96 wells in a 96 tissue culture plate), a first circuitboard includes 96 circular arrays containing 1 LED radially andsymmetrically distributed on each of the 96 circular arrays. In somecases, the method includes illuminating 24 independent channels, whereineach channel controls 4 wells of the 96 well plate. In some cases, theillumination device includes four LED drivers, each containing 24channels. In such cases, the four LED drivers, combined, include 96independent channels, wherein each channel controls one well of the 96wells in the tissue culture plate. In some cases, the four LED driversare chained together to provide independent control of each well in the96 wells of the tissue culture plate.

In some cases, the method includes reducing static or electricalshorting of the illumination device. In some cases, the method includesmounting the illumination device connected to the tissue culture plateonto a material through vibration-dampening mounts. A non-limitingexample of the material may include an acrylic, plastic, metal, orcomposite material or any material that is secure enough to constitute abase. In some cases, the acrylic material is an acrylic laser-cut base.In some cases, the vibration-dampening mounts include rubber footpegs.In some cases, the vibration-dampening mounts are configured to reducestatic or electrical shorting with the tissue culture incubation racks.

Light Guide Plates

Aspects of the present disclosure include contacting light from thelight source with one or more one or more light guide plates toilluminate the cell or the substrate in the one or more wells. In somecases, the light guide plates include one or more light guides. In somecases, contacting light with one or more light guides produces anarc-shaped light beam from the light source emitted by the light source.In some cases, the light beam from the light source includes 100 or moreμm, 200 or more μm, 300 or more μm, 400 or more μm, 500 or more μm, or600 or more μm arc of light. In some cases, the light beam from includes500 μm arc of light.

In some cases, the one or more light guide plates comprises a firstlight guide plate and a second light guide plate. In some cases, thefirst light guide and the second light guide can include light guidesthat are the same form of each other (e.g. made from the same material,and/or have the same dimensions, etc.). In some cases, the first lightguide and the second light guide can include light guides that aredifferent forms of each other (e.g. made from different materials,and/or have different dimensions, etc.)

In some cases, the one or more light guide plates include one or morelight guides. In some cases, the one or more light guides comprises 24light guides, 96 light guides, or 384 light guides. In some cases, theillumination device has one or more light guides within each of the oneor more light guide plates. For example, in some cases, the light guideswithin each of the one or more light guide plates are held in an arrayby a surrounding frame of the light guide plate. In some cases, theillumination device has 24 light guides held in an array by asurrounding frame of the light guide plate. In some cases, theillumination device has 96 light guides held in an array by asurrounding frame of the light guide plate. In some cases, theillumination device has 384 light guides held in an array by asurrounding frame of the light guide plate.

In some cases, the one or more light guides has a diameter of about 5 ormore mm, about 6 or more mm, about 7 or more mm, about 8 or more mm,about 9 or more mm, about 10 or more mm, about 11 or more mm, about 12or more mm, about 13 mm or more, about 15 mm or more, about 16 mm ormore, about 17 mm or more about 18 mm or more, about 19 mm or more, orabout 20 mm or more. In some cases, the one or more light guides has adiameter of about 7 or more mm. In some cases, the one or more lightguides has a diameter of about 16.5 or more mm. In some cases, the oneor more light guides has a radius of about 8.25 mm. In some cases, theone or more light guides has a diameter of about 16.5 mm. In some cases,the one or more light guides has a thickness ranging from 0.5 cm toabout 5 cm. In some cases, the one or more light guides has a thicknessof about 0.5 cm, about 1 cm, about 1.5 cm, about 2 cm, about 2.5 cm,about 3 cm, about 3.5 cm, about 4 cm, about 4.5 cm, or about 5 cm. Insome cases, the one or more light guides has a thickness ranging from 1cm to 1.5 cm. In some cases, the one or more light guides has athickness of about 1.5 cm.

In some cases, the one or more light guides can be of a circular shapeand/or other shapes as required per conditions specific to its intendeduse. In some cases, the one or more light guides comprises a circularshape. In some cases, the one or more light guides comprises acylindrical shape, a circular shape, a square shape, a spherical shape,a cone-shape, a prism-shape, or a rectangular shape. In some cases, eachof the light guides is the same shape. In some cases, each of the lightguides is a different shape. The light guides are not limited to theshapes and/or sizes as described herein and can be any shape and/or sizeas required per conditions specified to its intended use.

In some cases, the one or more light guide plates and/or light guides istransparent and made from a material selected from glass, acryl,plastic, polymethylmethacrylate (PMMA), poly-lactic acid (PLA), andepoxy. In some cases, the one or more light guide plates and/or lightguides is opaque except for the region of the light guide through whichlight passes through (e.g. the center of the light guide). In somecases, the one or more light guide plates and/or light guides istransparent in the center of the light guide through which light passesthrough.

In some cases, the one or more light guide plates is made from apolymer. In some cases, the polymer is acrylic or PLA. In some cases,the one or more light guides includes a reflective coating.

In some cases, the one or more light guides is configured to directlyreceive only light that is generated from the one or more LEDspositioned in the one or more circular arrays of the circuit board. Insome cases, each of one or more light guides is positioned to providefor selective illumination for each of the one or more wells of thetissue culture plate.

In some cases, the method includes positioning a first light guide plateadjacent (e.g. connected to) to the circuit board (e.g. the firstcircuit board or the second circuit board).

In some cases, the illumination uniformity of the light beam from lightemitted by the light source is proportional to the thickness of the oneor more light guides. In some cases, increasing the thickness of thelight guide decreases the difference between the edge and centerintensities of the one or more wells. In some cases, increasing thethickness of the light guide from 1 cm to 1.5 cm decreases differencebetween the edge and center intensities of the one or more wells. Insome cases, the one or more light guides improves the illuminationuniformity by about 10% or more, 20% or more, 30% or more, 40% or more,50% or more, 60% or more, or 70% or more as compared to an illuminationdevice without a light guide.

In some cases, the light guide is configured to decrease the variabilityof light intensity of the light beam emitted from the light sourcebetween each of the one or more wells of the tissue culture plate.

Optical Diffusers

In some aspects, the illumination device includes one or more opticaldiffusers. In some cases, the one or more optical diffusers comprises afirst optical diffuser and a second optical diffuser. In some cases, thefirst light guide plate is positioned between the first and secondoptical diffuser. However, optical elements beside diffusers can also beused to collimate the light to make the light beam more uniform. Anon-limiting example of an optical element other than a diffuserincludes Fresnel lenses, which is a lens that takes the shape of a flatsheet and can be used instead of a diffuser to collimate the light.

In some cases, the one or more optical diffusers comprises one or more80° circular optical diffusers. Optical diffusers can be any materialthat diffuses or scatters light. In some cases, the one or more opticaldiffusers and light guides control the uniformity and intensity of thesignals being projected onto the cell or substrate. In some cases, theone or more diffusers include one or more 80° diffuser coatings. (e.g.scatter coating, full width-half max). In some cases, the diffusercoating is coated on a polycarbonate material. In some cases, thepolycarbonate has a thickness of about 0.1 inches. However, the one ormore diffusers are not limited to 80° diffuser coatings and can be anyknown diffuser coating applied to different substrates.

In some cases, the first optical diffuser is positioned between thetissue culture plate and the first light guide plate. In some cases, thesecond optical diffuser is positioned between the first light guideplate and the second light guide plate. In some cases, the first opticaldiffuser is positioned between the tissue culture plate and the firstlight guide plate. In some cases, the second optical diffuser ispositioned between the first light guide plate and the second lightguide plate. In some cases, the first optical diffuser and the secondoptical diffuser can include optical diffusers that are the same form ofeach other (e.g. made from the same material, and/or have the samedimensions, etc.). In some cases, the first optical diffuser and thesecond optical diffuser can include optical diffusers that are differentforms of each other (e.g. made from different materials, and/or havedifferent dimensions, etc.)

Optical Masks

In some cases, the method includes selectively blocking a wavelength oflight outside of a surface (e.g. core region, aperture, cut-out feature,slit, etc.) of one or more optical masks. In some cases, the methodincludes selectively blocking a wavelength of light outside of a coreregion of one or more optical masks. In some cases, the illuminationdevice includes one or more optical masks. In some cases, the one ormore optical masks are configured to selectively block a wavelength oflight outside of a core region of the one or more optical masks fromreaching a detector to detect the light. In some cases, the one or moreoptical masks is configured to block a wavelength of light from reachingthe illuminated sample or substrate.

In some cases, the one or more optical masks includes an aperture. Insome cases, the method includes cutting (e.g. removing, extracting,laser cutting, die-cutting, etching, puncturing, etc.) a portion of anoptical mask to obtain a core region. In some cases, the one or moreoptical masks includes one or more cut-out features. In some cases, theone or more cut-out features includes a patterned cut-out feature.

In some cases, the one or more patterned cut-out features includes acore region. In some cases, the core region includes a pattern sizeranging from 10-600 μm. In some cases, the core region includes apattern size ranging from 50-500 μm. In some cases, the core regionincludes a pattern size of about 50 or more μm, 100 or more μm, 150 ormore μm, 200 or more μm, 250 or more μm, 300 or more μm, 350 or more μm,400 or more μm, 450 or more μm, 500 or more μm, 550 or more μm, or 600or more μm.

In some cases, the one or more patterned cut-out features is a slit. Insome cases, the one or more patterned cut-out features is a curved slit(e.g., an arc). In some cases, the one or more patterned cut-outfeatures is a circle, rectangle, square, or triangle. In some cases, thepatterned cut-out feature can be of a slit shape and/or other shapes asrequired per conditions specific to its intended use.

In some cases, the method includes selectively blocking the passage oflight outside of the core region of the optical mask.

In some cases, the one or more optical masks are made of an opaquematerial. In some cases, the core region of the one or more optical maskdoes not include the opaque material from which the optical mask is madefrom (e.g. the core region includes an aperture or a patterned slit inthe material). In some cases, the one or more optical masks include acombination of a transparent material and an opaque material. In somecases, the combination of a transparent material and an opaque materialprovides for greyscale modulation of the light pattern. In some cases,the core region of the optical mask includes a transparent material, andthe material outside of the core region is an opaque material.

In some cases, the method includes adhering the one or more opticalmasks to the one or more wells of the tissue culture plate. In somecases, the method includes positioning the one or more optical masks ona surface of one or more wells of the tissue culture plate. In somecases, the method includes positioning the one or more optical masks ona surface of the one or more wells. In some cases, the method includespositioning the one or more optical masks on a bottom outer surface ofthe one or more wells. In some cases, the method includes positioning acore region of the one or more optical masks in the center of an outersurface of the one or more wells. In some cases, the method includespositioning the one or more optical masks on the one or more wells ofthe tissue culture plate. In some cases, the method includes positioningthe one or more optical masks on an outer surface of the coverglassbottom. In some cases, the method includes positioning the one or moreoptical masks at a distal end (e.g. the closed-ended outer surface ofthe one or more wells) of the one or more wells of the tissue cultureplate relative to an open ended surface of the one or more wells. Insome cases, the method includes positioning the one or more opticalmasks on a surface of one or more wells of the tissue culture plate.

In some cases, the method includes positioning the one or more opticalmasks includes on each of the one or more wells of the tissue cultureplate. In some cases, the one or more optical masks comprises 24 opticalmasks, each mask positioned on each of the 24 wells of the tissueculture plate. In some cases, the one or more optical masks includes anoptical mask positioned on each of the one or more wells of the tissueculture plate. In some cases, the one or more optical masks comprises 96optical masks, each mask positioned on each of the 96 wells of thetissue culture plate. In some cases, the one or more optical masksincludes an optical mask positioned on each of the one or more wells ofthe tissue culture plate. In some cases, the one or more optical maskscomprises 384 optical masks, each mask positioned on each of the 384wells of the tissue culture plate.

In some cases, the optical mask includes a photo mask or an intensitymask. A “photo mask”, as used herein in its conventional sense, refersto a material, coating, and/or plate with holes or transparencies thatallow light to pass through in a defined pattern. In some cases, thephoto mask absorbs light to varying degrees and can be patterned asrequired per conditions specific to its intended use. In some cases, thephoto mask is an intensity mask. In some cases, the intensity mask isfully absorbing (e.g. opaque, dark), or not absorbing (e.g. transparent,bright), or a combination thereof. In some cases, the intensity mask ismade from adhesive vinyl. In some cases, the adhesive vinyl is polyvinylchloride (PVC). In some cases, the intensity mask is made from BiaxiallyOriented Polypropylene.

In some cases, the optical mask is a phase mask. In some cases, thephase mask is a phase shift mask. A phase mask is used to modulate thephase of light in order to change the light intensity. The phasemodulation results in constructive and destructive interference thatgenerates a pattern of light intensity, which can be similar to a lightpattern generated with an intensity mask. It can be used in combinationwith an intensity mask, which is then called a phase-shift mask. In somecases, the phase mask is made from glass (e.g. quartz).

In some cases, the illumination pattern when the light beam contacts theone or more wells includes a 0.5 mm diameter of light, 1.0 mm diameterof light, 1.5 mm diameter of light, or a 2 mm diameter of light emittedto the one or more wells. In some cases, the illumination patternincludes a 1.5 mm diameter of light emitted to the one or more wells.

In some cases, the one or more wells includes one or more cells. In somecases, the cell migrates a distance beyond the boundary of the coreregion. In some cases, the distance is 50 μm or more beyond the boundaryof the core region. In some cases, the distance is 500 μm beyond theboundary of the core region.

Detector

In some aspects, the method includes detecting light emitted from thecell or substrate with one or more detectors. In some aspects, theillumination device can be used in combination with a microscope, aspectrophotometer, a detector, or a robotic handler. Non-limitingexamples of microscopes include a fluorescence microscope, a confocallaser scanning microscope, and/or a bright-field microscope), aspectrophotometer, or a detector. In some cases, the detector is aphotomultiplier tube, a charged coupled device (CCD), or a complementarymetal oxide semiconductor (CMOS) sensor. In some cases, the detectors ofinterest are configured to measure collected light at one or morewavelengths, such as at 2 or more wavelengths, such as at 5 or moredifferent wavelengths, such as at 10 or more different wavelengths, suchas at 25 or more different wavelengths, such as at 50 or more differentwavelengths, such as at 100 or more different wavelengths, such as at200 or more different wavelengths, such as at 300 or more differentwavelengths and including measuring light emitted by a sample in theflow stream at 400 or more different wavelengths. In some embodiments,the method includes measuring collected light over a range ofwavelengths (e.g., 200 nm to 1000 nm). In some embodiments, measuringincludes measuring absorbance, reflectance, emission intensity, and/orfluorescence of the light signals. In some embodiments, detectors areconfigured to collect spectra of light over a range of wavelengths. Insome embodiments, an optical imaging system may include one or moredetectors configured to collect spectra of light over one or more of thewavelength ranges of 200 nm to 1000 nm. In some embodiments, detectorsof are configured to measure light emitted by a cell or substrate in theone or more wells of a tissue culture plate at one or more specificwavelengths. For example, the one or more detectors are configured tomeasure light at one or more of 350 nm, 370 nm, 400 nm, 410 nm, 450 nm,518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm,667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm,617 nm and any combinations thereof. In some embodiments, one or moredetectors may be configured to be paired with specific fluorophores,such as those used in a fluorescence assay.

In some cases, the method includes positioning the one or more detectorsabout 5 or more mm away from the light source. In some cases, the methodincludes positioning the one or more detectors about 10 mm or more, 15mm or more, 20 mm or more, or 25 mm or more away from the light source.In some cases, the method includes positioning the one or more detectorsis about 21 mm away from the light source.

Graphical User Interface (GUI)

In some cases, the method includes controlling the illumination devicewith a graphical user interface (GUI) to communicate wirelessly with thecontroller of the illumination device. In some cases, the methodincludes programming, with the GUI, the illumination parameters of theone or more wells. In some cases, the GUI can be used to wirelesslyprogram the illumination intensity and temporal (e.g. time) sequences ofeach of the one or more wells. In some cases, the GUI provides for auser to input a desired illumination parameter for each of the one ormore wells. In some cases, the method includes wirelessly uploading theillumination parameters to the controller (e.g. the microcontrollerand/or the LED driver) on the illumination device through the GUI.

In some cases, the method includes modulating the illuminationparameters of the illumination device through the GUI. In some cases,the method includes setting each channel of the LED driver (e.g. in a 24channel LED driver or a 96-channel LED driver) with one or moreillumination parameters through the GUI. In some cases, the GUI allowsthe user to set each channel to, for example, constant illumination at aspecified intensity; blinking illumination at a specified intensity,duty cycle, and time period; and a series of linear or sinusoidalfunctions at specified illumination parameters.

In some cases, the GUI provides for multiple piecewise functions thatcan be programmed in a time sequence.

Illumination Parameters

In some cases, the method includes spatially and temporally controllinglight from the illumination device to spatially and temporally controlillumination of a cell or a substrate with one or more illuminationpatterns. The term “spatial control” as used herein, refers tocontrolling (e.g. modulating or varying) a spatial distribution of lightwaves in space (e.g. controlling the intensity, amplitude, frequency,and/or phase of the light waves emitted to a targeted area; controllingthe distance of the light beam; controlling the distribution of thelight beam). The term “temporal control” as used herein, refers tocontrolling (e.g. modulating or varying) a distribution (e.g. intensity,amplitude, frequency, and/or phase) of light waves over time.

In some cases, the illumination parameters can include, but are notlimited to, frequency of light emitted from the light source, dutycycle, duration of light emitted from the light source, and specificpatterns of illumination (e.g. pulsed illumination). In some cases, theone or more illumination parameters is selected from an illuminationintensity, an illumination duration, an illumination pattern, and acombination thereof.

In some cases, the illumination parameter is an amplitude of light. Insome cases, the illumination parameter is a light frequency. In somecases, the illumination parameter is a phase of light.

In some cases, the illumination intensity ranges from about 0.005 μW/mm²to about 20 μW/mm². In some cases, the illumination intensity rangesfrom about 0.005 μW/mm² to about 10 μW/mm². In some cases, theillumination intensity ranges from about 0.005 μW/mm² to about 20mW/mm².

In some cases, the method includes illuminating the cell or thesubstrate of the one or more wells of the tissue culture plate with anillumination pattern. In some cases, the illumination pattern is apulsing pattern, where light is pulsed in a millisecond time frame. Insome cases, an illumination duration ranges from about 0.5 or more ms, 1or more ms, 2 or more ms, 3 or more ms, 4 or more ms, 5 or more ms, 6 ormore ms, 7 or more ms, 8 or more ms, or 10 or more ms. In some cases,the pulsed illumination includes a pulse duration of 1 or more ms.

In some cases, the method includes illuminating the cell or thesubstrate with a pulsed illumination. In some cases, the illuminationpattern is a pulsing pattern, where light is pulsed in a millisecondtime frame. In some cases, the illumination device further includes apulse generator configured to pulse the light emitted from the lightsource.

In some cases, the illumination pattern includes a sinusoidal or linearpattern with a pulsing frequency of 1 or more Hz.

In some cases, the illumination pattern includes a blinking pattern witha pulsing frequency of about 1 or more Hz, 10 or more Hz, 20 or more Hz,30 or more Hz, 40 or more Hz, 50 or more Hz, 60 or more Hz, 70 or moreHz, 80 or more Hz, 90 or more Hz, 100 or more Hz, 100 or more Hz, 110 ormore Hz, 120 or more Hz, 130 or more Hz, 140 or more Hz, or 150 or moreHz. In some cases, the illumination pattern includes a blinking patternwith a pulsing frequency of about 100 Hz.

In some cases, the illumination pattern includes a 0.5 mm diameter oflight, 1.0 mm diameter of light, 1.5 mm diameter of light, or a 2 mmdiameter of light emitted to the one or more wells. In some cases, theillumination pattern includes a 1.5 mm diameter of light emitted to theone or more wells.

In some cases, the illumination duration includes illumination of theone or more wells for 1 or more hours. In some cases, the illuminationduration includes illumination of the one or more wells for 1 or moreweeks. In some cases, the illumination duration ranges from about 0.5 ormore ms, 1 or more ms, 2 or more ms, 3 or more ms, 4 or more ms, 5 ormore ms, 6 or more ms, 7 or more ms, 8 or more ms, or 10 or more ms. Insome cases, the illumination duration ranges from about 0.5 or more s, 1or more s, 2 or more s, 3 or more s, 4 or more s, 5 or more s, 6 or mores, 7 or more s, 8 or more s, or 10 or more s. In some cases, theillumination duration ranges from about 1 or more minutes, 2 or moreminutes, 3 or more minutes, 4 or more minutes, 5 or more minutes, 6 ormore minutes, 7 or more minutes, 8 or more minutes, or 10 or moreminutes.

In some cases, the one or more wells can be illuminated with red, amber,yellow, green, blue, or white light. In some cases, the GUI can set thecolor of light corresponding to a specific wavelength.

In some cases, the method includes modulating the wavelength through theGUI to allow the user to set a wavelength of light for which light canbe emitted. In some cases, the method includes illuminating,stimulating, and/or activating the cell or the substrate with a lighthaving a wavelength ranging from 200 to 1000 nm. In some cases, thelight can have a wavelength ranging from about 350 to about 410 nm. Insome cases, the light can have a wavelength of about 470 nm and about510 nm or can have a wavelength of about 490 nm. In some cases, thelight can have a wavelength of about 470 nm. In some cases, the lightcan have a wavelength of about 445 nm. In some cases, the light can havea wavelength ranging from about 530 to about 595 nm. In some cases, thelight can have a wavelength of about 530 nm. In some cases, the lightcan have a wavelength of about 560 nm. In some cases, the light can havea wavelength of about 542 nm. In some cases, the light can have awavelength of about 546 nm. In some cases, the light can have awavelength ranging from about 580 and 630 nm. In some cases, the lightcan be at a wavelength of about 589 nm or the light can have awavelength greater than about 630 nm (e.g. less than about 740 nm). Inanother embodiment, the light has a wavelength of around 630 nm.

EXAMPLES OF NON-LIMITING ASPECTS OF THE DISCLOSURE

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-30 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

1. A system for spatially and temporally controlling light, the systemcomprising:

a tissue culture plate comprising one or more wells, wherein the one ormore wells comprises a cell or a substrate; an illumination devicepositioned adjacent to the tissue culture plate, wherein theillumination device comprises: a light source operably connected to acircuit board and configured to produce light; one or more light guideplates comprising one or more light guides; one or more optical maskspositioned adjacent to the one or more wells of the tissue cultureplate; a controller; a computer readable medium, comprising instructionsthat, when executed by the controller, cause the controller to:illuminate the cell or the substrate in the one or more wells with lightfrom the light source; and spatially and temporally control illuminationof the cell or the substrate with one or more illumination parameters,wherein the one or more light guides is configured to provide uniformillumination of the light in the one or more wells of the tissue cultureplate.

2. The system of Aspect 1, wherein the cell is a stem cell or aprogenitor cell.

3. The system of Aspect 1, wherein the system further comprises amicroscope, a spectrophotometer, or a detector.

4. The system of Aspect 1, wherein the one or more optical masks is aphoto mask, intensity mask, or a phase mask.

5. The system of Aspect 1, wherein the one or more optical masks ispositioned on a bottom surface of the one or more wells of the tissueculture plate.

6. The system of Aspect 1, wherein the illumination device furthercomprises one or more optical diffusers.

7. The system of Aspect 1, wherein the one or more illuminationparameters is selected from an illumination intensity, an illuminationduration, and an illumination pattern, and a combination thereof.

8. The system of Aspect 1, wherein the system further comprises agraphical user interface (GUI) configured to communicate wirelessly withthe controller.

9. The system of Aspect 1, wherein the one or more optical maskscomprises one or more patterned cut-out features comprising a coreregion.

10. The system of Aspect 9, wherein the core region comprises a patternsize ranging from 50-600 μm.

11. The system of Aspect 9, wherein the one or more optical masks isconfigured to selectively block the passage of light outside of the coreregion.

12. The system of Aspect 9, wherein the core region of the one or moreoptical masks is positioned in the center of an outer surface of the oneor more wells.

13. The system of Aspect 1, wherein the light source comprises one ormore light-emitting diodes (LEDs).

14. The system of Aspect 13, wherein the one or more wells isindependently illuminated by the one or more LEDs.

15. The system of Aspect 13, wherein the one or more LEDs issymmetrically and radially distributed on one or more circular arrays onthe circuit board.

16. The system of Aspect 15, wherein the circuit board comprises 5 LEDssymmetrically and radially distributed on each of the one or morecircular arrays of the circuit board.

17. The system of Aspect 15, wherein each of the one or more circulararrays has a radius of about 5 mm.

18. The system of Aspect 15, wherein the one or more light guides isconfigured to directly receive only light that is generated from the oneor more LEDs positioned in the one or more circular arrays of thecircuit board.

19. The system of Aspect 1, wherein the illumination uniformity of thelight beam from light emitted by the light source is proportional to thethickness of the one or more light guides.

20. The system of Aspect 1, wherein the light guide is configured todecrease the variability of light intensity of the light beam emittedfrom the light source between each of the one or more wells of thetissue culture plate.

21. An illumination device for spatially and temporally controllinglight, the device comprising: an illumination device connected to atissue culture plate comprising a cell or a substrate in one or morewells of the tissue culture plate, wherein the illumination devicecomprises: a light source operably adjacent to a circuit board andconfigured to produce light; one or more light guide plates comprisingone or more light guides; one or more optical masks positioned on asurface of the one or more wells of the tissue culture plate; acontroller; a computer readable medium, comprising instructions that,when executed by the controller, cause the controller to: illuminate thecell or the substrate with light from the light source; and spatiallyand temporally control illumination of light to the cell or thesubstrate in the one or more wells with one or more illuminationparameters, wherein the one or more light guides is configured toprovide uniform illumination of the light in the one or more wells ofthe tissue culture plate.

22. The device of Aspect 21, wherein the one or more light guides has athickness ranging from 0.5 cm to 5 cm.

23. The device of Aspect 21, further comprising one or more opticaldiffusers.

24. The device of Aspect 21, further comprising a heat sink mounted onthe circuit board.

25. The device of Aspect 21, further comprising a cooling fan.

26. The device of Aspect 21, wherein the one or more illuminationparameters is selected from an illumination intensity, an illuminationduration, an illumination pattern, and a combination thereof.

27. The device of Aspect 21, wherein the illumination uniformity of thelight beam from light emitted by the light source is proportional to thethickness of the one or more light guides.

28. The device of Aspect 21, wherein the light guide is configured todecrease the variability of light intensity of the light beam emittedfrom the light source between each of the one or more wells of thetissue culture plate.

29. A method for selectively spatially and temporally controlling light,the method comprising: activating a cell or a substrate with light,wherein the cell or the substrate is within one or more wells of atissue culture plate, wherein the light is generated by a systemcomprising: a light source operably connected to a circuit board andconfigured to produce light; one or more light guide plates comprisingone or more light guides; one or more optical masks positioned on asurface of the one or more wells of the tissue culture plate; acontroller; a computer readable medium, comprising instructions that,when executed by the controller, cause the controller to: illuminate thecell or the substrate in the one or more wells with the light from thelight source; and spatially and temporally control illumination of thecell or the substrate in the one or more wells with one or moreillumination parameters, wherein the one or more light guides isconfigured to provide uniform illumination of the light in the one ormore wells of the tissue culture plate.

30. The method of Aspect 29, wherein the cell is a stem cell or aprogenitor cell.

31. The method of Aspect 30, wherein the method is configured tospatially and temporally control stem cell or progenitor cell signalingand differentiation.

32. The method of Aspect 29, wherein the method further comprisesscreening for phototoxicity of the cell in response to light.

33. The method of Aspect 29, wherein the method further comprisesscreening for a candidate agent to determine whether the candidate agentmodulates an activity of the cell.

34. The method of Aspect 29, wherein the cell expresses alight-sensitive protein.

35. The method of Aspect 34, wherein the method further comprisesscreening for the fluorescent protein expressed in the cell in responseto light.

36. The method of Aspect 29, wherein the method is configured tophotopolymerize the substrate.

37. The method of Aspect 29, wherein the substrate is a polymer.

38. The method of Aspect 29, wherein the substrate is a hydrogel.

39. The method of Aspect 38, wherein the hydrogel comprises one or moremethacrylate groups.

40. The method of Aspect 39, wherein the hydrogel further comprises aphotoinitiator.

41. The method of Aspect 41, wherein the photoinitiator is lithiumphenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

42. The method of Aspect 38, wherein said activating provides forphotopatterning of the hydrogel in response to the light-activatablesignal.

43. The method of Aspect 40, wherein said activating provides forcrosslinkage of a first methacrylate group and a second methacrylategroup within the hydrogel.

44. The method of Aspect 38, wherein the method is configured tomodulate the stiffness of the hydrogel in response to light.

45. The method of Aspect 44, wherein the stiffness of the hydrogel isproportional to the light intensity of the light emitted by the lightsource.

46. The method of Aspect 44, wherein the hydrogel comprises one or morecells.

47. The method of Aspect 46, wherein the method further comprisesscreening for interactions of the one or more cell with an extracellularmatrix within the hydrogel.

48. The method of Aspect 29, wherein the illumination uniformity of thelight beam from light emitted by the light source is proportional to thethickness of the one or more light guides.

49. The method of Aspect 29, wherein the light guide is configured todecrease the variability of light intensity of the light beam emittedfrom the light source between each of the one or more wells of thetissue culture plate.

UTILITY

Example applications of the methods, devices, and systems of the presentdisclosure include use in high-throughput assays, such ashigh-throughput illumination and simultaneous recordings of varioussubstrates or samples (e.g. light-responsive bacterial or mammaliancells grown in tissue culture, hydrogels, dyes) with user-definedpatterns. The illumination device of the present disclosure can becombined with a robotic handler, a microscope, a spectrophotometer, orother conventional light detector to measure absorption, imagefluorescence, or optical signals from the sample. Additionalapplications include high-throughput screens, directed evolution oflight sensors and fluorescent proteins, phototoxicity screens,photopatterning of hydrogels, and cell signaling and differentiation.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Illumination Device for Spatial and Temporal Control ofMorphogen Signaling in Embryonic Stem Cell Cultures

Morphogen gradients are present throughout development and orchestratethe dynamic, coordinated movement and differentiation of cellpopulations. Spatially and temporally varying patterns of morphogenslocalize signaling to specific subpopulations of cells. Geneticperturbation and biomolecular treatment with pathway agonists orinhibitors have given immense insight into the key regulators ofdevelopmental progression, yet spatially-varying interactions betweencell subpopulations and time-varying signal dynamics and thresholdsremain largely unstudied, since such patterns of signaling are difficultto perturb and control in model developmental systems.

Light-responsive proteins from plants or bacteria have been adapted tocontrol signaling and protein interactions in mammalian cells, enablingthe optical techniques to stimulate signaling in a specific location, ata specific time. Following the discovery of light-gated ion channels, agreat variety of light-responsive domains have been discovered,optimized, and repurposed for optogenetic control of cell signaling.Using the heterodimerizing system iLID 3 for optogenetic control ofdevelopmental ERK signaling, the spatiotemporal limits of ERK have beenelucidated in the Drosophila embryo. Also in Drosophila, optogeneticclustering with Cryptochrome was used to inhibit Bcd transcription andWnt signaling, or activate migration through Rho signaling or cellcontractility. Optogenetics has also been applied to zebrafishdevelopment, for light-induced transcriptional activation of Nodaltarget genes and Rac-mediated cell migration.

Canonical Wnt/B-catenin signaling under optogenetic control using theillumination device of the present provided for mesendodermdifferentiation and cell migration in embryonic stem cells.

The present disclosure provides a robust, programmable illuminationsystem that can be easily incorporated into the workflow of routinetissue culture and allow spatial and temporal control of lightintensity.

Design

Programmable Illumination Devices for hESC Optogenetic Stimulation

To enable precise control over the intensity, timing, and location ofoptoWnt stimulation in hESC cultures, illumination devices wereengineered that incorporate into the workflow of stem cell culture,termed LAVA devices (light activation at variable amplitude; e.g.illumination device of the present disclosure). (FIG. 1A). LAVA devicesproject user-defined light patterns onto 24-well or 96-well tissueculture plate kept inside a tissue culture incubator (FIGS. 1B-1C, FIG.6). In brief, light from LEDs passes through optical elements thatensure uniform illumination of the multiwell plate. For stimulation ofoptoWnt, blue LEDs emitting at a central wavelength of 470 nm was chosento match the Cry2 absorption spectrum (FIG. 7), though LEDs of othercolors can be easily substituted for use with other optogenetic systems.An electronics subsystem allows programmed control of illuminationintensity and temporal sequences, with independent control of each well.Spatial precision is conveyed through an intensity mask attached to theculture plate. The hardware design also includes a cooling system andvibration isolation to ensure cell viability. Lastly, for ease of use, agraphical user interface (GUI) was developed to wirelessly program theillumination intensity and temporal sequences for each well (FIGS.8A-8B). A detailed protocol was provided for LAVA assembly, as well asdesign considerations, characterization, and proof of conceptapplications in spatiotemporal control of Wnt activation.

Optimization and Characterization of Illumination Uniformity

The LAVA optical system design was established by modeling LAVA in theoptical ray tracing software Zemax OpticStudio and optimizing foruniform well illumination (FIG. 2A). For simplicity and compactness,optical diffusers and scattering from the 3D-printed light guides wereused to improve illumination uniformity instead of lenses. In the Zemaxmodel, parameters such as LED position on the circuit board, diffuserstrength, and light guide dimensions were optimized to reduce intensitydrop-off at the edges of the tissue culture well (FIGS. 4A-4D, FIGS.5A-5E). Modeling results showed that the parameter with strongest effecton uniformity was the axial thickness, d, of the two 3D-printed lightguides (labelled in FIG. 1B). Based on the modeling results, LAVAdevices were fabricated and experimentally verified well uniformity byimaging LAVA wells under a low-magnification microscope (FIG. 2B).Measurement of light intensity as a function of radial distanceconfirmed relatively uniform illumination which improved with increasingd at the expense of maximum light intensity. Increasing d from 1 cm to1.5 cm improved the intensity decrease at the well edge from 20.4% to16.9%, i.e. a roughly 20% improvement in uniformity. A larger d alsoresulted in a two-fold improvement in well-to-well variability betweenthe 24 independent wells (2.6% versus 1.2% coefficient of variation)(FIG. 11). Considering there are many experimental applications whereintensity of illumination is paramount to well uniformity, the twohardware configurations for LAVA can be summarized as follows: (1) alow-intensity, high-precision configuration at d=1.5 cm where intensitycan be programmed 0 to 10 μW/mm² in 0.0024 μW/mm2 increments withimproved illumination uniformity and well-to-well variability and (2) ahigh-intensity, low precision configuration at d=1 cm where wellintensity resolution, variability, and uniformity are sacrificed for adoubling in light intensity (FIG. 2C).

Results Intensity Control of Optogenetic Stimulation Reveals BrachyuryExpression Level is Dependent on LRP6 Oligomer Number and Size

During mammalian embryonic development, gradients in Wnt signalintensity control the progression of cell lineage commitment and axispatterning. In hESCs, the intensity of Wnt signaling similarly modulatescell lineage commitment and differentiation potential. Equipped with amethod for optogenetic stimulation of cell cultures, LAVA and apreviously-established clonal optoWnt knock-in line was used foractivation of canonical Wnt signaling in hESCs. In contrast to simpleon/off control of Wnt signaling.

Since Cry2 oligomerization is a dynamic, reversible process whereclustering is triggered upon photon absorption, it was hypothesized thatthe fraction of ‘activated’ Cry2 proteins in a cell can be controlledwith light intensity (FIG. 3A). Indeed, the number and size of visibleLRP6 oligomers in a cell increased monotonically with light intensity(FIG. 3B). To determine whether increased Cry2 oligomerizationtranslated to a stronger Wnt/β-catenin signal intensity, expression ofBrachyury (Bra, also known by its gene name, T) was probed, a target ofWnt signaling and regulator of mesendoderm and primitive streakdifferentiation. Following a similar trend to Cry2 oligomerization, themean intensity of Bra immunostaining increased with light intensity,showing that an increased number and size of LRP6 clusters induces astronger differentiation signal (FIG. 3C, FIG. 12A).

To better quantify the variability of Bra expression at a single-celllevel, a clonal hESC cell line co-expressing the optoWnt system and aneGFP reporter was generated for endogenous Bra expression. Live-cellanalysis with flow cytometry showed a heterogeneous response to Wntstimulation at low light intensities (median, Q) and a more homogenous,higher eGFP expression at higher intensities (median, Q) (FIG. 12B).Maximal light-induced activation increased Bra expression by ˜33-foldover unilluminated optoWnt hESCs, which notably showed no detectableactivation in the dark. Quantification of the percentage eGFP-positivecells showed an exponential increase with light intensity that greatlyexceeded activation achieved with high concentrations of pathway agonistWnt3a (FIG. 3D). Fitting to an increasing exponential decay functionshowed a very rapid increase in Bra expression at low light intensities(time constant τ=0.07, see methods), with saturation reached at ˜0.4μW/mm2 The high sensitivity at lower light intensities shows a binaryswitch for onset of Bra expression above a signaling threshold, followedby a monotonic increase in Bra expression levels in a lightdose-dependent manner.

Analysis of Phototoxicity Reveals No Detectable Effects on hESC HealthBelow a 1 μW/Mm2 Illumination Intensity Threshold

Since high intensity light can induce phototoxicity in cells, thephototoxicity threshold was analyzed for hESC cultures using theillumination devices of the present disclosure. Continuous operation ofhigh-intensity LEDs can cause heating at high intensities (FIG. 13A).For all optogenetic stimulation an intensity range of under 1 μW/mm² wasused (specifically, 0.8 μW/mm2) Under this condition, no decrease inpluripotency markers Sox2, Nanog, and Oct4 were observed after 48 hrs ofillumination of wild-type hESCs, as well as no spontaneous Bra+mesendoderm differentiation (FIG. 13D). Notably, since saturation ofpercentage Bra+ optoWnt cells occurred at ˜0.4 μW/mm², the operatingrange of optoWnt stimulation falls below the 1 μW/mm2 phototoxicitythreshold.

Temporal Control of optoWnt Shows that Sustained Wnt Signaling isNecessary for Bra Activation

In addition to intensity control, LAVA enables temporal control ofillumination patterns (FIG. 4A). Oscillatory Wnt signals regulatemesoderm segmentation while local pulses of Wnt signaling are presentduring primitive streak patterning and neural tube development. Giventhe reversible oligomerization of Cry2, it was concluded that theoptoWnt system can be readily applied to studying temporal dynamics ofWnt signaling.

A LAVA GUI was designed to allow users to input the desired temporallight pattern for each well. The GUI wirelessly communicates with theLAVA board to set the illumination patterns for the duration of theexperiment. The user can set each of the 24 channels to one of threemodes: (1) constant illumination at a specified intensity; (2) blinkingat a specified intensity, duty cycle, and period; and (3) a series oflinear or sinusoidal functions at specified function parameters.Multiple piecewise functions can be programmed in sequence, enabling avariety of complex temporal light patterns (FIG. 4B). The shortestpossible blink, i.e. the temporal resolution of the device, was set to 1ms in firmware, though it was observed a significant drop in theaccuracy and precision of stimulation at pulsewidths below 10 ms (FIG.4C, FIG. 14)

The next step was to determine whether sustained Wnt activation isrequired for hESC mesendoderm differentiation. With the presence ofautoregulatory feedback loops and the potential for sustained Wntsignaling following a 24 hr pulse of CHIR treatment in mESC gastruloids,it sought to determine whether sustained Wnt activation is necessary forBra expression in hESCs, or whether shorter activation durations aresufficient for inducing a differentiation program. OptoWnt culturesexpressing a T/eGFP reporter were illuminated with varying durations oflight and quantified eGFP fluorescence with flow cytometry. Afterwithdrawal of illumination, T/eGFP levels decreased showing thatsustained illumination and optoWnt activation is necessary for asustained Bra transcriptional response in our hESC culture system.

Spatial Localization of Wnt Signaling and hESC Differentiation as aModel for Early Embryonic Wnt Patterning

An additional advantage of optogenetic control is the ability tomanipulate the spatial location of signal activation (FIG. 5, Panel A).Though precise patterned illumination can be achieved with confocalscanning or the use of spatial light modulators, the cost and complexityof such microscope systems are restrictive when low-resolution lightpatterns are sufficient. To more easily incorporate spatial lightpatterning during cell culture, die-cut intensity masks were designedthat can be adhered to the tissue culture plate during illumination(FIG. 15A). The mask feature size was limited by the cutting resolutionof the die cutter to ˜150 μm (FIG. 15B). In this way, wells of the24-well plate were illuminated with arbitrary light patterns and induceoptoWnt clustering only in illuminated regions (FIG. 5B). Since the maskis placed underneath the tissue culture plate, it was anticipated thatthere would be light scattering through the plate bottom (170 μm-thickcoverglass) that would compromise the mask resolution (FIG. 15C). Toquantify the extent of light scattering, the number of LRP6 clusters wasmeasured as a function of distance beyond the mask edge and found thatclusters were induced ˜50 μm from the mask edge, corresponding to a ˜100μm patterning resolution (see methods for calculation) (FIG. 5C).

During development, cells migrating through the primitive streak breakaway from the epithelial cell layer, scattering as single cells andadopting a mesenchymal morphology that initiates spatial patterning ofthe epiblast. To visualize the interaction between epithelial cells anddifferentiating mesendoderm, the spatial light patterning capability ofLAVA was used to illuminate optoWnt hESC cultures with an arc of light(FIG. 4D). Cells in the illuminated arc broke away from hESC colonies,adopted a mesenchymal morphology, and migrated up to ˜500 μm beyond theboundary of the light pattern. Given the 100 μm resolution ofpatterning, the possibility that cells were activated by lightscattering outside of the mask pattern was excluded. Immunostainingconfirmed that these migratory cells expressed Bra, while surroundingunilluminated cells retained epithelial morphology with no detectableBra expression. In addition, migratory cells showed a decrease in Oct4expression, a shift in β-catenin localization away from the plasmamembrane, and an increase in Slug expression, all consistent with cellsundergoing an epithelial-to-mesenchymal transition.

Illumination with a 1.5 mm-diameter circle of light allowed to betterquantify migration outside the area of illumination (FIG. 5F). Whenconfined by a higher seeding density, illuminated cells grew verticallyupwards, stacking 4 to 6 cell diameters in height (˜70 μm). Axialslicing with confocal imaging showed that cells in the illuminatedregion stained positive for Bra and migrated up to ˜300 μm outside ofthe illuminated region by diving underneath the epithelial cell layer.Taken together, these data show that optogenetic Wnt activation issufficient for inducing a migratory cell phenotype and that patternedillumination can be used as a tool to further study Wnt patterning andgastrulation-like events in culture.

DISCUSSION

Optogenetic control of Wnt signaling, optoWnt, and the LAVA system wereused to dynamically control morphogen signaling in hESCs. Throughextensive characterization of the LAVA board light patterning, precisemanipulation of stem cell signaling was demonstrated to be achieved inspace and time. Applications for LAVA devices can be extended to otherbiological systems and signal pathways for mechanistic spatiotemporalstudies or high-throughput optogenetic screens.

Design of LAVA Boards for Quantitative Control of Cell Signaling

A number of challenges exist for robust, long-term illumination of cellcultures. Cell toxicity from photodamage or overheating is a significantdrawback during optogenetic stimulation and can cause cell death ornon-specific activation of signaling pathways. Illumination uniformityacross a region of interest is critical as well, since optogeneticsignaling is dependent on light dosage (FIG. 3D). Because of this, aminimal intensity drop-off toward edges of the well is essential foruniform optogenetic activation within a well.

The illumination device of the present disclosure allows defined andhigh-throughput control of optogenetic signaling in cell cultures. Indesigning the LAVA boards, the optical system was optimized for uniformwell illumination (<17 drop-off) and designed electronics that enable16-bit control of intensity for 24 independent channels with arbitrarytemporal patterns (1 ms resolution) and spatial patterns through use ofa photomask (100 μm resolution). In this manner, two versions of theLAVA boards were that can illuminate 24-well or 96-well tissue cultureplates. Heating and vibration control were incorporated, characterizedthe phototoxicity threshold for hESCs under continuous illumination, anddeveloped a user interface to easily upload desired intensity patternsfor each channel to LAVA devices kept in a 37° C. tissue cultureincubator.

Efficiency and Dose-Responsiveness of Optogenetic Wnt/B-CateninSignaling

OptoWnt stimulation using the LAVA devices had high efficiency of Cry2clustering and Bra expression in hESCs. Following 24 hrs of continuousillumination at 0.4 μW/mm2, Bra expression was evident in >98% cells, acell percentage comparable to CHIR treatment and higher than Wnt3atreatment (FIG. 3D). Given that optoWnt modulates signaling at a nodehigh in the Wnt signaling cascade, i.e. at the Wnt-specific co-receptorLRP6, this high optoWnt efficiency is a significant advantage—optoWnt isthus as efficient as the commonly used CHIR small-molecule agonist withthe added advantage of high Wnt pathway specificity given the potentialnon-specific effects of GSK3B inhibition.

Additionally, the strength of optoWnt pathway activation wasdose-responsive to light intensity. The size and number of detectableLRP6c clusters per hESC increased with light intensity and correlatedwith increased Bra expression (FIG. 3B). This is likely due to anincreased fraction of photoactivated Cry2 molecules at higherillumination intensities and suggests a role for LRP6 cluster size orabundance in regulating B-catenin destruction complex dissociation 38 orsaturation. Analysis of the single-cell expression profiles in responseto varying light doses also elucidated signaling thresholds for Wntpathway activation. While the percentage of Bra+ cells increased sharplywith light intensity and saturates at 0.4 μW/mm2, Bra expression levelsincreased more gradually and saturate at ˜0.8 μW/mm2, which suggests abinary switch for the onset of Bra expression above a certain signalingthreshold followed by a light dose-dependent increase in expressionlevel (FIG. 3D; FIG. 12A). Such signaling thresholds can be furtherstudied through super-resolution imaging and quantification of clusterproperties. This operating range for optoWnt activation (0-0.8 μW/mm²)falls below the measured phototoxicity threshold of 1 μW/mm² (FIG. 13).

Spatial and Temporal Control of Wnt Signaling Dynamics

The LAVA boards and optoWnt system open a wide range of possibilitiesfor studying the role of Wnt dynamics in ESC signaling. Thereversibility of Cry2 clustering and Bra expression (FIG. 4D) combinedwith ease of temporal pattern generation with LAVA boards enablesintricate studies of Wnt signaling thresholds and timing of signalingoscillations during development.

The resolution of LAVA board spatial patterning and spatially localizedmesendoderm differentiation were quantified and used to mimic the Wntmorphogen gradients present in the early mammalian embryo. Patternedillumination allows for studying how the shape, size, and intensity ofspatial patterns influences differentiation and morphogenesis.

Materials & Methods LAVA Device Construction

LAVA devices are constructed using two custom printed circuit boards(PCB) designed in EAGLE (Autodesk). PCB1 contains electronics for LEDcontrol while PCB2 is the power distribution board. For 24-wellillumination, PCB1 contains solder pads for a circular array of 5 LEDsper well, which are connected in series and illuminate each well throughtwo 3D-printed light guides and a series of diffusers (opticalconfiguration optimized in Zemax, see below). For 96-well illumination,PCB1 contains solder pads for 1 LED per well of a 96-well plate; giventhe 24-channel LED driver, independent illumination control is possiblefor each group of 4 wells. For each channel, the ground wire connects toTLC5947 driver and is modulated with pulse-width modulation, while thepositive terminal connects to the power plane of PCB1. PCB1 alsocontains headers for electrical connection to cooling fans. A heatsinkmounts onto the bottom of PCB1, using thermally conductive adhesive(Arctic Silver, ASTA-7G), in a region without silk screen and thermallyconductive electrical vias that draw heat away from surface-mount LEDs.

In PCB2, a power supply connects through a barrel power jack to powerthe LEDs through an LED driver (TLC5947, Adafruit). Power is alsosupplied to three fans and the Raspberry Pi microcontroller throughswitching voltage regulators.

On top of PCB1, optical assembly and tissue culture plate is mounted insuch a way that tissue culture plate is illuminated from the bottom. Itis critical that tissue culture plate is made of black, opaque plastic athin, 170 μm coverslip bottom (Eppendorf Cell Imaging Plate, 24-well) toavoid light bleed-through between wells and high spatial patterningresolution. The LED driver, PCB2, and the Raspberry Pi microcontrollerare all mounted and electrically connected to PCB1, and the entireassembly is mounted onto an acrylic laser-cut base throughvibration-dampening mounts. The base contains rubber footpegs to reducestatic or electrical shorting with the tissue culture incubator racks.

Zemax Modeling and Optimization of LAVA Well

The ray-tracing software Zemax OpticStudio was used in Non-Sequentialmode to model illumination of a 24-well plate. Based on modelingresults, the optimized configuration parameters are as follows: 5surface-mount LEDs are symmetrically radially distributed around a 5mm-radius circle; diameter of each light guide is 16.5 mm; one 80°circular optical diffusers placed between the two light guides, anotherplaced onto the top light guide (i.e. between light guide and tissueculture plate); thickness of each light guide is 1.5 cm; light guidesare manufactured from black 3D-printed acrylic.

Software Control and Graphical User Interface

The LEDs are controlled by an Adafruit 24-Channel 12-bit PWM LED driverwith an SLI interface to a Raspberry Pi Zero W. The 12-bit PWM resultsin LEDs that are capable of 4096 unique illumination levels over theirgiven operating range. For ease of use, a GUI has been written in Javaand is conveniently packed into an executable application file. Thisinterface allows for independent control of each of the 24 channels. Toaccommodate for the variety of experimental conditions, each LED can beprogrammed to a constant illumination, a blinking pattern, or a seriesof linear and sinusoidal patterns. Since each board has slightlydifferent intensity characteristics, the intensity to PWM calibrationparameters are input at runtime. Sinusoidal and linear functions areinterpolated at a frequency of 1 Hz whereas blinking patterns have beentested up to 100 Hz. Since the LED board's USB port may be inaccessibleduring certain experiments, it is possible to wirelessly upload newillumination settings from any WIFI capable computer.

Upon booting the Raspberry Pi, a C++ script executes, checking thedevice for previous illumination settings and resumes the patternedillumination. The Pi polls for new illumination settings every fewseconds, so the changes of a newly uploaded pattern will be reflectedwithout an additional reboot. It should be noted that the decision touse C++ was motivated by a desire to break through certain speedlimitations posed by an interpreted language's rate of execution. APython implementation was completed, but only the compiled C++ versionis able drive all 24 channels at the desired refresh rate.

FIGS. 1A-1C. Overview of illumination device, LAVA, for optogeneticstimulation of hESC cultures. FIG. 1A) Schematic of optogenetic typicalexperiment, where spatiotemporal control is conferred through patterningof light. FIG. 1B) Diagram of illumination device design. LEDsilluminate a tissue culture plate placed on top of device, with lightpassing through a series of two light guides, two optical diffusers, anda die-cut mask. LEDs are programmed through a Raspberry Pi and LEDdriver, and cooled with a heatsink and cooling fans. FIG. 1C) Image ofassembled device, with optical, cooling, and electronics subsystemshighlighted.

FIGS. 2A-2C. Optical design for illumination uniformity of tissueculture wells. FIG. 2A) Schematic of Zemax model used for systemoptimization. FIG. 2B) Brightfield images of well (left) and graph ofintensity linescans along indicated cross-sections (right) characterizethe intensity uniformity of the illumination device under twoconfigurations, where light guide thickness d is either (1) 1 cm, topgreen or (2) 1.5 cm, bottom purple. Percent decrease is calculatedbetween intensity at center of well and intensity at highlighted redpoint, which indicates location of well edge of a 24-well culture plate(average of 4 independent wells). Scale bar 2.5 mm FIG. 2C) Lightintensity (irradiance, μW/mm²) in response to the programmed duty cycleof the pulse-width modulation signal. Graph shows intensity measuredfrom each of the 24 channels, as well as the curve fitting to a linearregression model.

FIGS. 3A-3D. Optogenetic induction of Bra expression is light-doseresponsive. FIG. 3A) Schematic of optoWnt system. In the dark, Cry2molecules are diffuse, while light illumination induces clustering ofLRP6c, stabilizing β-catenin and transcription of target genes. FIG. 3B)Immunostaining for LRP6 (left) and quantification of cluster number perhESC in response to increasing light intensity after 1 hr illumination.Graph shows individual cell quantification (black dot) and violin plotof distribution (blue). Scale bar 25 μm. FIG. 3C) Immunostaining forBrachyury in response to increasing light intensity after 24 hrillumination or 3 μM CHIR treatment. Scale bar 25 μm. FIG. 3D) Flowcytometry of optoWnt hESCs expressing eGFP reporter for Bra in responseto light stimulation or treatment with Wnt pathway agonists (Wnt3arecombinant protein or CHIR). Graph shows percent eGFP positive cellsand nonlinear least squares fit to increasing exponential decay curve.Graph shows mean±1 s.d., n=3 replicates.

FIGS. 4A-4D. Characterization of temporal control using LAVA devices.FIG. 4A) Schematic of temporal light patterning. FIG. 4B) Well intensityas a function of time of various waveforms programmed through LAVA GUI.Programmed values shown in black, measured intensity in green. FIG. 4C)Error in measured pulsewidths relative to programmed pulsewidth. FIG.4D) OptoWnt hESCs were illuminated for varying lengths of time followedby flow cytometry as fixed endpoint. Graph shows histograms of eGFPreporter for endogenous Bra/T activity for each illumination condition.Cell count histograms normalized to total cells per condition (˜30,000cells).

FIGS. 5A-5F. OptoWnt induces epithelial to mesenchymal transition andprimitive streak-like behavior. FIG. 5A) Schematic of spatial lightpatterning. FIG. 5B) Stitched brightfield and fluorescence images ofOptoWnt hESCs illuminated with UC Berkeley (Cal) logo mask andimmunostained for LRP6. Clusters of LRP6 are observed in illuminatedregion (orange inset) but not in masked region (yellow inset). Scale bar100 μm (top), 1 mm (bottom). FIG. 5C) Quantification of light scatteringthrough bottom of tissue culture plate shows a ˜50 μm spread (full widthat half max, red line) of hESC OptoWnt clusters outside of projectedpattern (orange line). Brightfield image of mask (top), fluorescenceimage of immunostaining for LRP6 (middle), and quantification of LRP6cluster count (bottom). Insets (1) and (2) show masked, unclustered andilluminated, clustered regions, respectively. Scale bars 100 μm. FIG.5D) Patterned illumination with 500 μm arc of light. Brightfield image(left panel) with overlay of light pattern shows migratory cells withmesenchymal morphology outside of region of illumination (white arrows)Immunostaining for Bra and total β-catenin (middle panel) and Oct4 andSlug (right panel) with overlay of light pattern (yellow line).Greyscale zoom-in of highlighted region (white box) shows migratorycells. Scale bars 200 μm. FIG. 5E) Patterned illumination with 1.5 mmdiameter circle of light Immunostaining for Bra shows expression inregion of mask (left panel). Confocal z-stacks (middle panel) of bottomcell layer (Z=2.4 μm), middle (Z=20.8) and top (Z=47.2) show migratingcells diving beneath epithelial cell layer. Z-slice through alonghighlighted line (green). FIG. 5F) Quantification of cell migrationbeyond region of mask illumination in (FIG. 5E). Graph shows mean±1s.d., n=3 replicates. Student's t-test (two-tail). Scale bars 200 μm.

FIG. 6. System block diagram of LAVA device.

FIG. 7. Emission spectrum of 470 nm blue LEDs matches absorptionspectrum of Cry2.

FIGS. 8A-8B. Screenshot of GUI for illumination device control. User caninput parameters for desired intensities, blinking sequences, ortemporal functions for each individual well and upload settingswirelessly to the device.

FIGS. 9A-9H. Validation of Zemax ray tracing model. Schematic of LEDconfiguration (left), modeling result at detector plane (middle), andcolumn and row cross-sections (right) with well edge of 24-well plateindicated with red points. FIG. 9A) Single LED illuminating detector 21mm away. FIG. 9B) Five LEDs, distributed along 1 cm diameter circle,illuminating detector 21 mm away. FIG. 9C) Five LEDs illuminatingdetector 2 mm away. FIG. 9D) Five LEDs illuminating detector 21 mm awaythrough two 0.01″ thick sheets of polycarbonate. FIG. 9E) Five LEDsilluminating detector 21 mm away through two 10 mm transparent lightguides. FIG. 9F) Five LEDs illuminating detector 21 mm away through two0.01″ thick sheets of polycarbonate and two 10 mm transparent lightguides. FIG. 9G) Five LEDs illuminating detector 21 mm away through two0.01″ thick sheets of polycarbonate with 80° diffuser coating and two 10mm transparent light guides. FIG. 9H) Five LEDs illuminating detector 21mm away through two uncoated 0.01″ thick sheets of polycarbonate and two10 mm reflective light guides with Lambertian scattering.

FIGS. 10A-10E. Results of Zemax modeling at variable light guidethicknesses, d₁ and d₂. FIG. 10A) Schematic of modeling setup. Five LEDsilluminate detector through two 0.01″ thick sheets of polycarbonate with80° diffuser coating (red) and two reflective light guides withLambertian scattering (grey cylinder). FIGS. 10B-10E) Modeling resultsat indicated values of d₁, d₂. Image at detector plane (left) and columnand row cross-sections (right) with well edge of 24-well plate indicatedwith red points show improved illumination uniformity at expense oflight intensity with increasing light guide thickness.

FIG. 11. Coefficient of variation of light intensity between the 24independent light channels measured at different programmed intensities.Green points correspond to optical configuration with d=1 cm, violetpoints show optical configuration with d=1.5 cm.

FIGS. 12A-12B. FIG. 12A) Immunostaining quantification for average Braintensity per hESC in response to increasing light intensity after 24 hrillumination or 3 μM CHIR treatment. Graph shows mean±1 s.d., n=3replicates. FIG. 12B) Flow cytometry histograms of optoWnt hESCsexpressing eGFP reporter for Bra after 24 hr illumination at varyinglight intensities. Graph shows sum of n=3 replicates.

FIGS. 13A-13D. Phototoxicity during continuous optogenetic stimulationof hESC cultures. FIG. 13A) Temperature of media after 24 hrs ofcontinuous illumination. FIG. 13B) Brightfield images (top) of livewild-type hESC cultures illuminated at indicated light intensities for48 hrs and flow cytometry results for Annexin V and propidium iodide(PI) stain. Scale bar 250 μm. FIG. 13C) Quantification of apoptosismarker Annexin V and dead cell stain PI shows a significant increase inapoptosis and cell death above 1 μW/mm² illumination intensity(p_(A)=0.002, p_(PI)=0.001 0-1 μW/mm² and p_(A)=0.0005, p_(PI)=0.00050-2 μW/mm²). No difference was observed between 0 and 0.5 μW/mm²(p_(A)=0.78, p_(PI)=0.50). ANOVA followed by Tukey test. Graph showsmean±1 s.d., n=3 replicates. FIG. 13D) Representative fluorescenceimages (left) and quantification (right) of wild-type hESCs stained forpluripotency markers Sox2, Nanog, Oct4, and differentiation markerBrachyury (Bra/T) after 48 hr illumination at 0.8 μW/mm². Student'st-test (two-tail). Graph shows mean±1 s.d., n=3 replicates. Scale bar 25μm.

FIG. 14. Illumination power meter measurements of programmed blinkingsequences show signal inaccuracy at 1 ms pulses. Voltage signal frompower meter measured with oscilloscope and is proportional toirradiance.

FIGS. 15A-15C. FIG. 15A) Images of adhesive die-cut masks applied usingtransfer tape (top) onto 24-well cell culture plate (bottom). FIG. 15B)Brightfield images of die-cut mask illustrate resolution limit ofcutter. Scale bar 3 mm FIG. 15C) Schematic of light scattering fromphotomask.

FIG. 16. Screenshot of Zemax model parameters of LAVA well, optimizedfor uniform 24-well illumination.

FIGS. 17A-17B. Circuit board layout (top) and schematic (bottom) for24-well LAVA device, PCB1.

FIGS. 18A-18B. Circuit board layout (top) and schematic (bottom) forLAVA device power distribution, PCB2.

FIGS. 19A-19B. Circuit board layout (top) and schematic (bottom) for96-well LAVA device, PCB1.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-49. (canceled)
 50. A system for spatially and temporally controllingstem cell or progenitor cell signaling, differentiation, or both, thesystem comprising: (a) a plurality of stem cells or progenitor cells;and (b) an illumination device positioned to illuminate the plurality ofstem cells or progenitor cells, the illumination device comprising: (i)a light source that produces light; and (ii) a controller, wherein thelight source illuminates the plurality of stem cells or progenitor cellswith light, wherein the controller spatially and temporally controlsillumination of the plurality of stem cells or progenitor cells with oneor more illumination patterns comprising one or more illuminationparameters, wherein the one or more illumination parameters comprisesillumination intensity, and wherein the system spatially and temporallycontrols stem cell or progenitor cell signaling, differentiation, orboth.
 51. The system of claim 50, wherein the one or more illuminationparameters further comprises one or more of: an illumination duration,an illumination pattern, an illumination wavelength, and any combinationthereof.
 52. The system of claim 50, wherein the light source comprisesone or more light-emitting diodes (LEDs).
 53. The system of claim 50,wherein at least one cell of the plurality of stem cells or progenitorcells is genetically engineered to express a light-activatable protein.54. The system of claim 53, wherein the light source illuminates theplurality of stem cells or progenitor cells with light at a wavelengthsufficient to activate the light-activatable protein in the at least onecell.
 55. The system of claim 53, wherein an amount of activatedlight-activatable protein is controlled by the illumination intensity.56. The system of claim 53, wherein the light-activatable protein isselected from the group consisting of: a cryptochrome, a photoactiveyellow protein (PYP) photosensor, a photoreceptor of blue light usingflavin adenine dinucleotide (BLUF), a light, oxygen or voltage sensing(LOV) domain, and a phytochrome.
 57. The system of claim 53, wherein thelight-activatable protein is a light-inducible dimerizer.
 58. The systemof claim 53, wherein the light-activatable dimerizer is selected fromthe group consisting of: a CRY2/CIB system, a Phy/Pif system, and aBphP1/PpsR2 system.
 59. The system of claim 50, wherein the illuminationdevice is configured to be placed into an incubator.
 60. The system ofclaim 50, wherein the illumination device is wirelessly controllable.61. The system of claim 50, further comprising one or more light guideplates comprising one or more light guides.
 62. The system of claim 61,wherein the one or more light guide plates is configured to provideuniform illumination of the light to at least a subset of the pluralityof stem cells or progenitor cells.
 63. The system of claim 50, furthercomprising one or more optical masks.
 64. The system of claim 63,wherein the one or more optical masks comprises a photo mask, anintensity mask, or a phase mask.
 65. The system of claim 50, wherein atleast one cell of the plurality of stem cells or progenitor cells isindependently illuminated by the light source.
 66. The system of claim50, wherein the plurality of stem cells or progenitor cells are on amulti-well plate.
 67. The system of claim 50, wherein the plurality ofstem cells or progenitor cells are on a tissue culture plate.
 68. Thesystem of claim 50, further comprising a graphical user interface (GUI)configured to communicate with the controller.
 69. The system of claim68, wherein the GUI is configured to communicate wirelessly with thecontroller.
 70. The system of claim 68, wherein the GUI is configured toallow a user to define the one or more illumination parameters.
 71. Thesystem of claim 50, wherein the system is configured to spatially andtemporally pattern differentiation of the plurality of stem cells orprogenitor cells.
 72. The system of claim 50, wherein the illuminationintensity is below an amount that causes phototoxicity in the pluralityof stem cells or progenitor cells.
 73. The system of claim 50, whereinthe illumination intensity is from about 0.005 μW/mm² to about 20μW/mm².
 74. The system of claim 50, further comprising a pulse generatorconfigured to pulse the light emitted from the light source.
 75. Thesystem of claim 50, wherein the one or more illumination patterns is apulsing pattern, a sinusoidal pattern, a linear pattern, a blinkingpattern, or any combination thereof.
 76. The system of claim 50, whereinthe one or more illumination parameters comprises an illuminationduration, and wherein the illumination duration is about 0.5 ms or more.77. The system of claim 50, wherein the one or more illuminationparameters comprises a pulsing frequency, and wherein the pulsingfrequency is about 1 Hz or more.
 78. The system of claim 50, wherein theone or more illumination parameters comprises an illumination duration,and wherein the illumination duration is about 1 minute or more.
 79. Thesystem of claim 50, wherein the light source is configured to illuminatea first subset of the plurality of stem cells or progenitor cells withlight at a first wavelength, and to illuminate a second subset of theplurality of stem cells or progenitor cells with light at a secondwavelength.